Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplications U.S. Serial No. 60/211,387 filed Jun. 14, 2000 (Atty.Docket CL000667-PROV) and U.S. Ser. No. 60/268,023, filed Feb. 13, 2001.

FIELD OF THE INVENTION

[0002] The present invention is in the field of transporter proteinsthat are related to the potassium channel transporter subfamily,recombinant DNA molecules, and protein production. The present inventionspecifically provides novel peptides and proteins that effect ligandtransport and nucleic acid molecules encoding such peptide and proteinmolecules, all of which are useful in the development of humantherapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0003] Transporters

[0004] Transporter proteins regulate many different functions of a cell,including cell proliferation, differentiation, and signaling processes,by regulating the flow of molecules such as ions and macromolecules,into and out of cells. Transporters are found in the plasma membranes ofvirtually every cell in eukaryotic organisms. Transporters mediate avariety of cellular functions including regulation of membranepotentials and absorption and secretion of molecules and ion across cellmembranes. When present in intracellular membranes of the Golgiapparatus and endocytic vesicles, transporters, such as chloridechannels, also regulate organelle pH. For a review, see Greger, R.(1988) Annu. Rev. Physiol. 50:111-122.

[0005] Transporters are generally classified by structure and the typeof mode of action. In addition, transporters are sometimes classified bythe molecule type that is transported, for example, sugar transporters,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of molecule (a detailed reviewof channel types can be found at Alexander, S. P. H. and J. A. Peters:Receptor and transporter nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 (1997) andhttp://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0006] The following general classification scheme is known in the artand is followed in the present discoveries.

[0007] Channel-type transporters. Transmembrane channel proteins of thisclass are ubiquitously found in the membranes of all types of organismsfrom bacteria to higher eukaryotes. Transport systems of this typecatalyze facilitated diffusion (by an energy-independent process) bypassage through a transmembrane aqueous pore or channel without evidencefor a carrier-mediated mechanism. These channel proteins usually consistlargely of a-helical spanners, although b-strands may also be presentand may even comprise the channel. However, outer membrane porin-typechannel proteins are excluded from this class and are instead includedin class 9.

[0008] Carrier-type transporters. Transport systems are included in thisclass if they utilize a carrier-mediated process to catalyze uniport (asingle species is transported by facilitated diffusion), antiport (twoor more species are transported in opposite directions in a tightlycoupled process, not coupled to a direct form of energy other thanchemiosmotic energy) and/or symport (two or more species are transportedtogether in the same direction in a tightly coupled process, not coupledto a direct form of energy other than chemiosmotic energy).

[0009] Pyrophosphate bond hydrolysis-driven active transporters.Transport systems are included in this class if they hydrolyzepyrophosphate or the terminal pyrophosphate bond in ATP or anothernucleoside triphosphate to drive the active uptake and/or extrusion of asolute or solutes. The transport protein may or may not be transientlyphosphorylated, but the substrate is not phosphorylated.

[0010] PEP-dependent, phosphoryl transfer-driven group translocators.Transport systems of the bacterial phosphoenolpyruvate:sugarphosphotransferase system are included in this class. The product of thereaction, derived from extracellular sugar, is a cytoplasmicsugar-phosphate.

[0011] Decarboxylation-driven active transporters. Transport systemsthat drive solute (e.g., ion) uptake or extrusion by decarboxylation ofa cytoplasmic substrate are included in this class.

[0012] Oxidoreduction-driven active transporters. Transport systems thatdrive transport of a solute (e.g., an ion) energized by the flow ofelectrons from a reduced substrate to an oxidized substrate are includedin this class.

[0013] Light-driven active transporters. Transport systems that utilizelight energy to drive transport of a solute (e.g., an ion) are includedin this class.

[0014] Mechanically-driven active transporters. Transport systems areincluded in this class if they drive movement of a cell or organelle byallowing the flow of ions (or other solutes) through the membrane downtheir electrochemical gradients.

[0015] Outer-membrane porins (of b-structure). These proteins formtransmembrane pores or channels that usually allow the energyindependent passage of solutes across a membrane. The transmembraneportions of these proteins consist exclusively of b-strands that form ab-barrel. These porin-type proteins are found in the outer membranes ofGram-negative bacteria, mitochondria and eukaryotic plastids.

[0016] Methyltransferase-driven active transporters. A singlecharacterized protein currently falls into this category, theNa+-transporting methyltetrahydromethanopterin:coenzyme Mmethyltransferase.

[0017] Non-ribosome-synthesized channel-forming peptides or peptide likemolecules. These molecules, usually chains of L- and D-amino acids aswell as other small molecular building blocks such as lactate, formoligomeric transmembrane ion channels. Voltage may induce channelformation by promoting assembly of the transmembrane channel. Thesepeptides are often made by bacteria and fungi as agents of biologicalwarfare.

[0018] Non-Proteinaceous Transport Complexes. Ion conducting substancesin biological membranes that do not consist of or are not derived fromproteins or peptides fall into this category.

[0019] Functionally characterized transporters for which sequence dataare lacking. Transporters of particular physiological significance willbe included in this category even though a family assignment cannot bemade.

[0020] Putative transporters in which no family member is an establishedtransporter. Putative transport protein families are grouped under thisnumber and will either be classified elsewhere when the transportfunction of a member becomes established, or will be eliminated from theTC classification system if the proposed transport function isdisproven. These families include a member or members for which atransport function has been suggested, but evidence for such a functionis not yet compelling.

[0021] Auxiliary transport proteins. Proteins that in some wayfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport are included in this class.These proteins always function in conjunction with one or more transportproteins. They may provide a function connected with energy coupling totransport, play a structural role in complex formation or serve aregulatory function.

[0022] Transporters of unknown classification. Transport proteinfamilies of unknown classification are grouped under this number andwill be classified elsewhere when the transport process and energycoupling mechanism are characterized. These families include at leastone member for which a transport function has been established, buteither the mode of transport or the energy coupling mechanism is notknown.

[0023] Ion channels

[0024] An important type of transporter is the ion channel. Ion channelsregulate many different cell proliferation, differentiation, andsignaling processes by regulating the flow of ions into and out ofcells. Ion channels are found in the plasma membranes of virtually everycell in eukaryotic organisms. Ion channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,ion channels, such as chloride channels, also regulate organelle pH. Fora review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0025] Ion channels are generally classified by structure and the typeof mode of action. For example, extracellular ligand gated channels(ELGs) are comprised of five polypeptide subunits, with each subunithaving 4 membrane spanning domains, and are activated by the binding ofan extracellular ligand to the channel. In addition, channels aresometimes classified by the ion type that is transported, for example,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of ion (a detailed review ofchannel types can be found at Alexander, S. P. H. and J. A. Peters(1997). Receptor and ion channel nomenclature supplement. TrendsPharmacol. Sci., Elsevier, pp. 65-68 andhttp://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0026] There are many types of ion channels based on structure. Forexample, many ion channels fall within one of the following groups:extracellular ligand-gated channels (ELG), intracellular ligand-gatedchannels (ILG), inward rectifying channels (INR), intercellular (gapjunction) channels, and voltage gated channels (VIC). There areadditionally recognized other channel families based on ion-typetransported, cellular location and drug sensitivity. Detailedinformation on each of these, their activity, ligand type, ion type,disease association, drugability, and other information pertinent to thepresent invention, is well known in the art.

[0027] Extracellular ligand-gated channels, ELGs, are generallycomprised of five polypeptide subunits, Unwin, N. (1993), Cell 72:31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J.Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem.239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), TrendsPharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J.Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions:this serves as a means of identifying other members of the ELG family ofproteins. ELG bind a ligand and in response modulate the flow of ions.Examples of ELG include most members of the neurotransmitter-receptorfamily of proteins, e.g., GABAI receptors. Other members of this familyof ion channels include glycine receptors, ryandyne receptors, andligand gated calcium channels.

[0028] The Voltage-gated Ion Channel (VIC) Superfamily

[0029] Proteins of the VIC family are ion-selective channel proteinsfound in a wide range of bacteria, archaea and eukaryotes Hille, B.(1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolutionand diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed.,Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993),Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci.,Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20:91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. andW. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo-or heterooligomeric structures with several dissimilar subunits (e.g.,a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), butthe channel and the primary receptor is usually associated with the a(or al) subunit. Functionally characterized members are specific for K⁺,Na⁺ or Ca²⁺. The K⁺ channels usually consist of homotetramericstructures with each a-subunit possessing six transmembrane spanners(TMSs). The al and a subunits of the Ca²⁺ and Na⁺ channels,respectively, are about four times as large and possess 4 units, eachwith 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs.These large channel proteins form heterotetra-unit structures equivalentto the homotetrameric structures of most K⁺ channels. All four units ofthe Ca²⁺ and Na⁺ channels are homologous to the single unit in thehomotetrameric K⁺ channels. Ion flux via the eukaryotic channels isgenerally controlled by the transmembrane electrical potential (hencethe designation, voltage-sensitive) although some are controlled byligand or receptor binding.

[0030] Several putative K⁺-selective channel proteins of the VIC familyhave been identified in prokaryotes. The structure of one of them, theKcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Åresolution. The protein possesses four identical subunits, each with twotransmembrane helices, arranged in the shape of an inverted teepee orcone. The cone cradles the “selectivity filter” P domain in its outerend. The narrow selectivity filter is only 12 Å long, whereas theremainder of the channel is wider and lined with hydrophobic residues. Alarge water-filled cavity and helix dipoles stabilize K⁺ in the pore.The selectivity filter has two bound K⁺ ions about 7.5 Å apart from eachother. Ion conduction is proposed to result from a balance ofelectrostatic attractive and repulsive forces.

[0031] In eukaryotes, each VIC family channel type has several subtypesbased on pharmacological and electrophysiological data. Thus, there arefive types of Ca²⁺ channels (L, N, P, Q and T). There are at least tentypes of K⁺ channels, each responding in different ways to differentstimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive[BK_(Ca), IK_(Ca) and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)].There are at least six types of Na⁺ channels (I, II, III, μ1, H1 andPN3). Tetrameric channels from both prokaryotic and eukaryotic organismsare known in which each a-subunit possesses 2 TMSs rather than 6, andthese two TMSs are homologous to TMSs 5 and 6 of the six TMS unit foundin the voltage-sensitive channel proteins. KcsA of S. lividans is anexample of such a 2 TMS channel protein. These channels may include theK_(Na) (Na⁺-activated) and K_(Vol) (cell volume-sensitive) K⁺ channels,as well as distantly related channels such as the Tok1 K⁺ channel ofyeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and theTREK-1 K⁺ channel of the mouse. Because of insufficient sequencesimilarity with proteins of the VIC family, inward rectifier K⁺ IRKchannels (ATP-regulated; G-protein-activated) which possess a P domainand two flanking TMSs are placed in a distinct family. However,substantial sequence similarity in the P region suggests that they arehomologous. The b, g and d subunits of VIC family members, when present,frequently play regulatory roles in channel activation/deactivation.

[0032] The Epithelial Na⁺ Channel (ENaC) Family

[0033] The ENaC family consists of over twenty-four sequenced proteins(Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H.Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G.Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997),Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273:9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger,J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are fromanimals with no recognizable homologues in other eukaryotes or bacteria.The vertebrate ENaC proteins from epithelial cells cluster tightlytogether on the phylogenetic tree: voltage-insensitive ENaC homologuesare also found in the brain. Eleven sequenced C. elegans proteins,including the degenerins, are distantly related to the vertebrateproteins as well as to each other. At least some of these proteins formpart of a mechano-transducing complex for touch sensitivity. Thehomologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the firstpeptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0034] Protein members of this family all exhibit the same apparenttopology, each with N- and C-termini on the inside of the cell, twoamphipathic transmembrane spanning segments, and a large extracellularloop. The extracellular domains contain numerous highly conservedcysteine residues. They are proposed to serve a receptor function.

[0035] Mammalian ENaC is important for the maintenance of Na⁺ balanceand the regulation of blood pressure. Three homologous ENaC subunits,alpha, beta, and gamma, have been shown to assemble to form the highlyNa⁺-selective channel. The stoichiometry of the three subunits isalpha₂, beta1, gamma1 in a heterotetrameric architecture.

[0036] The Glutamate-gated Ion Channel (GIC) Family of NeurotransmitterReceptors

[0037] Members of the GIC family are heteropentameric complexes in whicheach of the 5 subunits is of 800-1000 amino acyl residues in length(Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol.Sci., Elsevier, pp. 36-40). These subunits may span the membrane threeor five times as putative a-helices with the N-termini (theglutamate-binding domains) localized extracellularly and the C-terminilocalized cytoplasmically. They may be distantly related to theligand-gated ion channels, and if so, they may possess substantialb-structure in their transmembrane regions. However, homology betweenthese two families cannot be established on the basis of sequencecomparisons alone. The subunits fall into six subfamilies: a, b, g, d, eand z.

[0038] The GIC channels are divided into three types: (1)a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate-and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors.Subunits of the AMPA and kainate classes exhibit 35-40% identity witheach other while subunits of the NMDA receptors exhibit 22-24% identitywith the former subunits. They possess large N-terminal, extracellularglutamate-binding domains that are homologous to the periplasmicglutamine and glutamate receptors of ABC-type uptake permeases ofGram-negative bacteria. All known members of the GIC family are fromanimals. The different channel (receptor) types exhibit distinct ionselectivities and conductance properties. The NMDA-selective largeconductance channels are highly permeable to monovalent cations andCa²⁺. The AMPA- and kainate-selective ion channels are permeableprimarily to monovalent cations with only low permeability to Ca²⁺.

[0039] The Chloride Channel (ClC) Family

[0040] The ClC family is a large family consisting of dozens ofsequenced proteins derived from Gram-negative and Gram-positivebacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer,K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J.Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol.242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher,W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998),Annu. Rev. Physiol. 60: 689-717). These proteins are essentiallyubiquitous, although they are not encoded within genomes of Haemophilusinfluenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequencedproteins vary in size from 395 amino acyl residues (M. jannaschii) to988 residues (man). Several organisms contain multiple ClC familyparalogues. For example, Synechocystis has two paralogues, one of 451residues in length and the other of 899 residues. Arabidopsis thalianahas at least four sequenced paralogues, (775-792 residues), humans alsohave at least five paralogues (820-988 residues), and C. elegans alsohas at least five (810-950 residues). There are nine known members inmammals, and mutations in three of the corresponding genes cause humandiseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiaeonly have one ClC family member each. With the exception of the largerSynechocystis paralogue, all bacterial proteins are small (395-492residues) while all eukaryotic proteins are larger (687-988 residues).These proteins exhibit 10-12 putative transmembrane a-helical spanners(TMSs) and appear to be present in the membrane as homodimers. While onemember of the family, Torpedo ClC-O, has been reported to have twochannels, one per subunit, others are believed to have just one.

[0041] All functionally characterized members of the ClC familytransport chloride, some in a voltage-regulated process. These channelsserve a variety of physiological functions (cell volume regulation;membrane potential stabilization; signal transduction; transepithelialtransport, etc.). Different homologues in humans exhibit differing anionselectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductancesequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5channels and others exhibit outward rectifying currents with currentsonly at voltages more positive than +20 mV.

[0042] Animal Inward Rectifier K⁺ Channel (IRK-C) Family

[0043] IRK channels possess the “minimal channel-forming structure” withonly a P domain, characteristic of the channel proteins of the VICfamily, and two flanking transmembrane spanners (Shuck, M. E., et al.,(1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995),Am. J. Physiol. 268: H506-H51 1; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78:227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171).They may exist in the membrane as homo- or heterooligomers. They have agreater tendency to let K⁺ flow into the cell than out.Voltage-dependence may be regulated by external K⁺ , by internal Mg²⁺,by internal ATP and/or by G-proteins. The P domains of IRK channelsexhibit limited sequence similarity to those of the VIC family, but thissequence similarity is insufficient to establish homology. Inwardrectifiers play a role in setting cellular membrane potentials, and theclosing of these channels upon depolarization permits the occurrence oflong duration action potentials with a plateau phase. Inward rectifierslack the intrinsic voltage sensing helices found in VIC family channels.In a few cases, those of Kir1.1a and Kir6.2, for example, directinteraction with a member of the ABC superfamily has been proposed toconfer unique functional and regulatory properties to the heteromericcomplex, including sensitivity to ATP. The SUR1 sulfonylurea receptor(spQ09428) is the ABC protein that regulates the Kir6.2 channel inresponse to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 arethe cause of familial persistent hyperinsulinemic hypoglycemia ininfancy (PHHI), an autosomal recessive disorder characterized byunregulated insulin secretion in the pancreas.

[0044] ATP-gated Cation Channel (ACC) Family

[0045] Members of the ACC family (also called P2X receptors) respond toATP, a functional neurotransmitter released by exocytosis from manytypes of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8:474-483; Soto, F., M. Garcia-Guzman and W. Stüthmer (1997), J. Membr.Biol. 160: 91-100). They have been placed into seven groups (P2X₁-P2X₇)based on their pharmacological properties. These channels, whichfunction at neuron-neuron and neuron-smooth muscle junctions, may playroles in the control of blood pressure and pain sensation. They may alsofunction in lymphocyte and platelet physiology. They are found only inanimals.

[0046] The proteins of the ACC family are quite similar in sequence(>35% identity), but they possess 380-1000 amino acyl residues persubunit with variability in length localized primarily to the C-terminaldomains. They possess two transmembrane spanners, one about 30-50residues from their N-termini, the other near residues 320-340. Theextracellular receptor domains between these two spanners (of about 270residues) are well conserved with numerous conserved glycyl and cysteylresidues. The hydrophilic C-termini vary in length from 25 to 240residues. They resemble the topologically similar epithelial Na⁺ channel(ENaC) proteins in possessing (a) N- and C-termini localizedintracellularly, (b) two putative transmembrane spanners, (c) a largeextracellular loop domain, and (d) many conserved extracellular cysteylresidues. ACC family members are, however, not demonstrably homologouswith them. ACC channels are probably hetero- or homomultimers andtransport small monovalent cations (Me⁺). Some also transport Ca²⁺; afew also transport small metabolites.

[0047] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel(RIR-CaC) Family

[0048] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate(IP3)-sensitive Ca²⁺-release channels function in the release of Ca²⁺from intracellular storage sites in animal cells and thereby regulatevarious Ca²⁺-dependent physiological processes (Hasan, G. et al., (1992)Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem.269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487;Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors andChannels (A. G. Lee, ed.), JAI Press, Denver, Colo., pp 291-326;Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ryreceptors occur primarily in muscle cell sarcoplasmic reticular (SR)membranes, and IP3 receptors occur primarily in brain cell endoplasmicreticular (ER) membranes where they effect release of Ca²⁺ into thecytoplasm upon activation (opening) of the channel.

[0049] The Ry receptors are activated as a result of the activity ofdihydropyridine-sensitive Ca²⁺ channels. The latter are members of thevoltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitivechannels are present in the T-tubular systems of muscle tissues.

[0050] Ry receptors are homotetrameric complexes with each subunitexhibiting a molecular size of over 500,000 daltons (about 5,000 aminoacyl residues). They possess C-terminal domains with six putativetransmembrane a -helical spanners (TMSs). Putative pore-formingsequences occur between the fifth and sixth TMSs as suggested formembers of the VIC family. The large N-terminal hydrophilic domains andthe small C-terminal hydrophilic domains are localized to the cytoplasm.Low resolution 3-dimensional structural data are available. Mammalspossess at least three isoforms that probably arose by gene duplicationand divergence before divergence of the mammalian species. Homologuesare present in humans and Caenorabditis elegans.

[0051] IP₃ receptors resemble Ry receptors in many respects. (1) Theyare homotetrameric complexes with each subunit exhibiting a molecularsize of over 300,000 daltons (about 2,700 amino acyl residues). (2) Theypossess C-terminal channel domains that are homologous to those of theRy receptors. (3) The channel domains possess six putative TMSs and aputative channel lining region between TMSs 5 and 6. (4) Both the largeN-terminal domains and the smaller C-terminal tails face the cytoplasm.(5) They possess covalently linked carbohydrate on extracytoplasmicloops of the channel domains. (6) They have three currently recognizedisoforms (types 1, 2, and 3) in mammals which are subject todifferential regulation and have different tissue distributions.

[0052] IP₃ receptors possess three domains: N-terminal IP₃-bindingdomains, central coupling or regulatory domains and C-terminal channeldomains. Channels are activated by IP₃ binding, and like the Ryreceptors, the activities of the IP₃ receptor channels are regulated byphosphorylation of the regulatory domains, catalyzed by various proteinkinases. They predominate in the endoplasmic reticular membranes ofvarious cell types in the brain but have also been found in the plasmamembranes of some nerve cells derived from a variety of tissues.

[0053] The channel domains of the Ry and IP₃ receptors comprise acoherent family that in spite of apparent structural similarities, donot show appreciable sequence similarity of the proteins of the VICfamily. The Ry receptors and the IP₃ receptors cluster separately on theRIR-CaC family tree. They both have homologues in Drosophila. Based onthe phylogenetic tree for the family, the family probably evolved in thefollowing sequence: (1) A gene duplication event occurred that gave riseto Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved frominvertebrates. (3) The three isoforms of each receptor arose as a resultof two distinct gene duplication events. (4) These isoforms weretransmitted to mammals before divergence of the mammalian species.

[0054] The Organellar Chloride Channel (O-ClC) Family

[0055] Proteins of the O-ClC family are voltage-sensitive chloridechannels found in intracellular membranes but not the plasma membranesof animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268:14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272:12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272:23880-23886).

[0056] They are found in human nuclear membranes, and the bovine proteintargets to the microsomes, but not the plasma membrane, when expressedin Xenopus laevis oocytes. These proteins are thought to function in theregulation of the membrane potential and in transepithelial ionabsorption and secretion in the kidney. They possess two putativetransmembrane a-helical spanners (TMSs) with cytoplasmic N- andC-termini and a large luminal loop that may be glycosylated. The bovineprotein is 437 amino acyl residues in length and has the two putativeTMSs at positions 223-239 and 367-385. The human nuclear protein is muchsmaller (241 residues). A C. elegans homologue is 260 residues long.

[0057] Transporter proteins, particularly members of the potassiumchannel subfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown transport proteins. Thepresent invention advances the state of the art by providing previouslyunidentified human transport proteins.

SUMMARY OF THE INVENTION

[0058] The present invention is based in part on the identification ofamino acid sequences of human transporter peptides and proteins that arerelated to the potassium channel transporter subfamily, as well asallelic variants and other mammalian orthologs thereof. These uniquepeptide sequences, and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulatetransporter activity in cells and tissues that express the transporter.Experimental data as provided in FIG. 1 indicates expression in humansin normal nervous tissue, brain, brain neuroblastoma, heart, kidney,lung, spleen, testis, and leukocyte.

DESCRIPTION OF THE FIGURE SHEETS

[0059]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the transporter protein of the presentinvention. (SEQ ID NO:1) In addition structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in humansin normal nervous tissue, brain, brain neuroblastoma, heart, kidney,lung, spleen, testis, and leukocyte.

[0060]FIG. 2 provides the predicted amino acid sequence of thetransporter of the present invention. (SEQ ID NO:2) In additionstructure and functional information such as protein family, function,and modification sites is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

[0061]FIG. 3 provides genomic sequences that span the gene encoding thetransporter protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 79 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0062] General Description

[0063] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a transporter protein or part of atransporter protein and are related to the potassium channel transportersubfamily. Utilizing these sequences, additional genomic sequences wereassembled and transcript and/or cDNA sequences were isolated andcharacterized. Based on this analysis, the present invention providesamino acid sequences of human transporter peptides and proteins that arerelated to the potassium channel transporter subfamily, nucleic acidsequences in the form of transcript sequences, cDNA sequences and/orgenomic sequences that encode these transporter peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thetransporter of the present invention.

[0064] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known transporter proteins ofthe potassium channel transporter subfamily and the expression patternobserved. Experimental data as provided in FIG. 1 indicates expressionin humans in normal nervous tissue, brain, brain neuroblastoma, heart,kidney, lung, spleen, testis, and leukocyte. The art has clearlyestablished the commercial importance of members of this family ofproteins and proteins that have expression patterns similar to that ofthe present gene. Some of the more specific features of the peptides ofthe present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown potassium channel family or subfamily of transporter proteins.

[0065] Specific Embodiments

[0066] Peptide Molecules

[0067] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetransporter family of proteins and are related to the potassium channeltransporter subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIGS. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the transporter peptides of the present invention,transporter peptides, or peptides/proteins of the present invention.

[0068] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprising theamino acid sequences of the transporter peptides disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

[0069] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0070] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0071] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetransporter peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0072] The isolated transporter peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in normal nervous tissue, brain, brain neuroblastoma, heart,kidney, lung, spleen, testis, and leukocyte. For example, a nucleic acidmolecule encoding the transporter peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0073] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0074] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0075] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the transporter peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0076] The transporter peptides of the present invention can be attachedto heterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a transporter peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the transporter peptide. “Operativelylinked” indicates that the transporter peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the transporter peptide.

[0077] In some uses, the fusion protein does not affect the activity ofthe transporter peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant transporter peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0078] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al, Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A transporter peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the transporter peptide.

[0079] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0080] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the transporter peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0081] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0082] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New. Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch (JMol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

[0083] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0084] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the transporter peptides of the present invention as well asbeing encoded by the same genetic locus as the transporter peptideprovided herein. As indicated by the data presented in FIG. 3, the mapposition was determined to be on chromosome 9 by ePCR, and confirmedwith radiation hybrid mapping.

[0085] Allelic variants of a transporter peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the transporterpeptide as well as being encoded by the same genetic locus as thetransporter peptide provided herein. Genetic locus can readily bedetermined based on the genomic information provided in FIG. 3, such asthe genomic sequence mapped to the reference human. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 9 by ePCR, and confirmed with radiation hybrid mapping. Asused herein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a transporter peptide encoding nucleic acid moleculeunder stringent conditions as more fully described below.

[0086]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 79 different nucleotide positions, includingnon-synonymous coding SNPs at positions 26367, 32860, and 39908. Changesin the amino acid sequence caused by these SNPs is indicated in FIG. 3and can readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a reference. Some of of theseSNPs that are located outside the ORF and in introns may affect genetranscription. Positioning of each SNP in an exon, intron, or outsidethe ORF can readily be determined using the DNA position given for eachSNP and the start/stop, exon, and intron genomic coordinates given inFIG. 3.

[0087] Paralogs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a transporter peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

[0088] Orthologs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

[0089] Non-naturally occurring variants of the transporter peptides ofthe present invention can readily be generated using recombinanttechniques. Such variants include, but are not limited to deletions,additions and substitutions in the amino acid sequence of thetransporter peptide. For example, one class of substitutions areconserved amino acid substitution. Such substitutions are those thatsubstitute a given amino acid in a transporter peptide by another aminoacid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0090] Variant transporter peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind ligand, abilityto transport ligand, ability to mediate signaling, etc. Fully functionalvariants typically contain only conservative variation or variation innon-critical residues or in non-critical regions. FIG. 2 provides theresult of protein analysis and can be used to identify criticaldomains/regions. Functional variants can also contain substitution ofsimilar amino acids that result in no change or an insignificant changein function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

[0091] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0092] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al, Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as transporter activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0093] The present invention further provides fragments of thetransporter peptides, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

[0094] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a transporter peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the transporter peptide or could be chosenfor the ability to perform a function, e.g. bind a substrate or act asan immunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe transporter peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0095] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally intransporter peptides are described in basic texts, detailed monographs,and the research literature, and they are well known to those of skillin the art (some of these features are identified in FIG. 2).

[0096] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0097] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N. Y Acad. Sci. 663:48-62(1992)).

[0098] Accordingly, the transporter peptides of the present inventionalso encompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature transporter peptide is fused withanother compound, such as a compound to increase the half-life of thetransporter peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature transporter peptide, suchas a leader or secretory sequence or a sequence for purification of themature transporter peptide or a pro-protein sequence.

[0099] Protein/Peptide Uses

[0100] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in atransporter-effector protein interaction or transporter-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0101] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0102] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, transporters isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in normal nervous tissue,brain, brain neuroblastoma, heart, kidney, lung, spleen, testis, andleukocyte. Specifically, a virtual northern blot shows expression innormal nervous tissue, adult brain, and brain neuroblastoma. Inaddition, PCR-based tissue screening panels indicate expression inbrain, heart, kidney, lung, spleen, testis, and leukocyte. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of transporter proteins, particularly members of thepotassium channel subfamily (see Background of the Invention). Thestructural and functional information provided in the Background andFigures provide specific and substantial uses for the molecules of thepresent invention, particularly in combination with the expressioninformation provided in FIG. 1. Experimental data as provided in FIG. 1indicates expression in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte. Suchuses can readily be determined using the information provided herein,that known in the art and routine experimentation.

[0103] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to transporters that arerelated to members of the potassium channel subfamily. Such assaysinvolve any of the known transporter functions or activities orproperties useful for diagnosis and treatment of transporter-relatedconditions that are specific for the subfamily of transporters that theone of the present invention belongs to, particularly in cells andtissues that express the transporter. Experimental data as provided inFIG. 1 indicates that the transporter proteins of the present inventionare expressed in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.Specifically, a virtual northern blot shows expression in normal nervoustissue, adult brain, and brain neuroblastoma. In addition, PCR-basedtissue screening panels indicate expression in brain, heart, kidney,lung, spleen, testis, and leukocyte. The proteins of the presentinvention are also useful in drug screening assays, in cell-based orcell-free systems ((Hodgson, Bio/technology, 1992, Sept 10(9);973-80).Cell-based systems can be native, i.e., cells that normally express thetransporter, as a biopsy or expanded in cell culture. Experimental dataas provided in FIG. 1 indicates expression in humans in normal nervoustissue, brain, brain neuroblastoma, heart, kidney, lung, spleen, testis,and leukocyte. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the transporter protein.

[0104] The polypeptides can be used to identify compounds that modulatetransporter activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thetransporter. Both the transporters of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thetransporter. These compounds can be further screened against afunctional transporter to determine the effect of the compound on thetransporter activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thetransporter to a desired degree.

[0105] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the transporter protein and a molecule that normally interactswith the transporter protein, e.g. a substrate or a component of thesignal pathway that the transporter protein normally interacts (forexample, another transporter). Such assays typically include the stepsof combining the transporter protein with a candidate compound underconditions that allow the transporter protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the transporter protein and thetarget, such as any of the associated effects of signal transductionsuch as changes in membrane potential, protein phosphorylation, cAMPturnover, and adenylate cyclase activation, etc.

[0106] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al, Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0107] One candidate compound is a soluble fragment of the receptor thatcompetes for ligand binding. Other candidate compounds include mutanttransporters or appropriate fragments containing mutations that affecttransporter function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

[0108] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) transporter activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate transporter activity. Thus, the transport of aligand, change in cell membrane potential, activation of a protein, achange in the expression of genes that are up- or down-regulated inresponse to the transporter protein dependent signal cascade can beassayed.

[0109] Any of the biological or biochemical functions mediated by thetransporter can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the transporter can be assayed.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in normalnervous tissue, brain, brain neuroblastoma, heart, kidney, lung, spleen,testis, and leukocyte. Specifically, a virtual northern blot showsexpression in normal nervous tissue, adult brain, and brainneuroblastoma. In addition, PCR-based tissue screening panels indicateexpression in brain, heart, kidney, lung, spleen, testis, and leukocyte.

[0110] Binding and/or activating compounds can also be screened by usingchimeric transporter proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a ligand-binding region can be usedthat interacts with a different ligand then that which is recognized bythe native transporter. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the transporter is derived.

[0111] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the transporter (e.g. binding partners and/orligands). Thus, a compound is exposed to a transporter polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble transporter polypeptide is also added to themixture. If the test compound interacts with the soluble transporterpolypeptide, it decreases the amount of complex formed or activity fromthe transporter target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the transporter. Thus, the soluble polypeptide that competes with thetarget transporter region is designed to contain peptide sequencescorresponding to the region of interest.

[0112] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the transporter protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

[0113] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of transporter-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a transporter-binding protein and a candidate compound are incubatedin the transporter protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the transporter protein target molecule, or which arereactive with transporter protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0114] Agents that modulate one of the transporters of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0115] Modulators of transporter protein activity identified accordingto these drug screening assays can be used to treat a subject with adisorder mediated by the transporter pathway, by treating cells ortissues that express the transporter. Experimental data as provided inFIG. 1 indicates expression in humans in normal nervous tissue, brain,brain neuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.These methods of treatment include the steps of administering amodulator of transporter activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

[0116] In yet another aspect of the invention, the transporter proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the transporter and are involved in transporteractivity. Such transporter-binding proteins are also likely to beinvolved in the propagation of signals by the transporter proteins ortransporter targets as, for example, downstream elements of atransporter-mediated signaling pathway. Alternatively, suchtransporter-binding proteins are likely to be transporter inhibitors.

[0117] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a transporterprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming atransporter-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the transporter protein.

[0118] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a transporter-modulating agent, an antisensetransporter nucleic acid molecule, a transporter-specific antibody, or atransporter-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0119] The transporter proteins of the present invention are also usefulto provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates expression in humans in normal nervoustissue, brain, brain neuroblastoma, heart, kidney, lung, spleen, testis,and leukocyte. The method involves contacting a biological sample with acompound capable of interacting with the transporter protein such thatthe interaction can be detected. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

[0120] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0121] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered transporter activity incell-based or cell-free assay, alteration in ligand or antibody-bindingpattern, altered isoelectric point, direct amino acid sequencing, andany other of the known assay techniques useful for detecting mutationsin a protein. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0122] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0123] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol Physiol. 23(10-1 1):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the transporter protein in which oneor more of the transporter functions in one population is different fromthose in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other ligand-binding regions thatare more or less active in ligand binding, and transporter activation.Accordingly, ligand dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

[0124] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.Accordingly, methods for treatment include the use of the transporterprotein or fragments.

[0125] Antibodies

[0126] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0127] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0128] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0129] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0130] Antibodies are preferably prepared from regions or discretefragments of the transporter proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/ortransporter/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0131] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0132] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0133] Antibody Uses

[0134] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the transporter proteins of the present invention are expressed inhumans in normal nervous tissue, brain, brain neuroblastoma, heart,kidney, lung, spleen, testis, and leukocyte. Specifically, a virtualnorthern blot shows expression in normal nervous tissue, adult brain,and brain neuroblastoma. In addition, PCR-based tissue screening panelsindicate expression in brain, heart, kidney, lung, spleen, testis, andleukocyte. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full length protein can beused to identify turnover.

[0135] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0136] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin normal nervous tissue, brain, brain neuroblastoma, heart, kidney,lung, spleen, testis, and leukocyte. The diagnostic uses can be applied,not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0137] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0138] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in normalnervous tissue, brain, brain neuroblastoma, heart, kidney, lung, spleen,testis, and leukocyte. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0139] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the transporter peptide to abinding partner such as a ligand or protein binding partner. These usescan also be applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

[0140] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0141] Nucleic Acid Molecules

[0142] The present invention further provides isolated nucleic acidmolecules that encode a transporter peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the transporter peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0143] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0144] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0145] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0146] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0147] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0148] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can compriseseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0149] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0150] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0151] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the transporter peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0152] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0153] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the transporter proteinsof the present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0154] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0155] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0156] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0157] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 9 by ePCR, and confirmed with radiation hybrid mapping.

[0158]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 79 different nucleotide positions, includingnon-synonymous coding SNPs at positions 26367, 32860, and 39908. Changesin the amino acid sequence caused by these SNPs is indicated in FIG. 3and can readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a reference. Some of of theseSNPs that are located outside the ORF and in introns may affect genetranscription. Positioning of each SNP in an exon, intron, or outsidethe ORF can readily be determined using the DNA position given for eachSNP and the start/stop, exon, and intron genomic coordinates given inFIG. 3.

[0159] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0160] Nucleic Acid Molecule Uses

[0161] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 79 differentnucleotide positions.

[0162] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0163] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0164] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0165] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0166] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome9 by ePCR, and confirmed with radiation hybrid mapping.

[0167] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0168] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0169] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0170] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0171] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0172] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the transporter proteins of the present invention areexpressed in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.Specifically, a virtual northern blot shows expression in normal nervoustissue, adult brain, and brain neuroblastoma. In addition, PCR-basedtissue screening panels indicate expression in brain, heart, kidney,lung, spleen, testis, and leukocyte.

[0173] Accordingly, the probes can be used to detect the presence of, orto determine levels of, a specific nucleic acid molecule in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression and/or gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in transporterprotein expression relative to normal results.

[0174] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0175] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a transporter protein, such asby measuring a level of a transporter-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if atransporter gene has been mutated. Experimental data as provided in FIG.1 indicates that the transporter proteins of the present invention areexpressed in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.Specifically, a virtual northern blot shows expression in normal nervoustissue, adult brain, and brain neuroblastoma. In addition, PCR-basedtissue screening panels indicate expression in brain, heart, kidney,lung, spleen, testis, and leukocyte.

[0176] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate transporter nucleic acid expression.

[0177] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the transporter gene, particularly biological andpathological processes that are mediated by the transporter in cells andtissues that express it. Experimental data as provided in FIG. 1indicates expression in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte. Themethod typically includes assaying the ability of the compound tomodulate the expression of the transporter nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired transporter nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the transporter nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

[0178] The assay for transporter nucleic acid expression can involvedirect assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thetransporter protein signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

[0179] Thus, modulators of transporter gene expression can be identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of mRNA determined. The level of expression oftransporter mRNA in the presence of the candidate compound is comparedto the level of expression of transporter mRNA in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of nucleic acid expression based on this comparison and beused, for example to treat a disorder characterized by aberrant nucleicacid expression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

[0180] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate transporter nucleic acidexpression in cells and tissues that express the transporter.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in normalnervous tissue, brain, brain neuroblastoma, heart, kidney, lung, spleen,testis, and leukocyte. Specifically, a virtual northern blot showsexpression in normal nervous tissue, adult brain, and brainneuroblastoma. In addition, PCR-based tissue screening panels indicateexpression in brain, heart, kidney, lung, spleen, testis, and leukocyte.Modulation includes both up-regulation (i.e. activation or agonization)or down-regulation (suppression or antagonization) or nucleic acidexpression.

[0181] Alternatively, a modulator for transporter nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the transporter nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in humans in normal nervous tissue, brain, brainneuroblastoma, heart, kidney, lung, spleen, testis, and leukocyte.

[0182] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe transporter gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0183] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in transporter nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in transporter genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the transporter gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the transporter geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a transporterprotein.

[0184] Individuals carrying mutations in the transporter gene can bedetected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in the gene encodingthe transporter protein of the present invention. SNPs were identifiedat 79 different nucleotide positions, including non-synonymous codingSNPs at positions 26367, 32860, and 39908. Changes in the amino acidsequence caused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of of these SNPs that arelocated outside the ORF and in introns may affect gene transcription.Positioning of each SNP in an exon, intron, or outside the ORF canreadily be determined using the DNA position given for each SNP and thestart/stop, exon, and intron genomic coordinates given in FIG. 3. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 9 by ePCR, and confirmed with radiationhybrid mapping. Genomic DNA can be analyzed directly or can be amplifiedby using PCR prior to analysis. RNA or cDNA can be used in the same way.In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al, Nucleic AcidsRes. 23:675-682 (1995)). This method can include the steps of collectinga sample of cells from a patient, isolating nucleic acid (e.g., genomic,mRNA or both) from the cells of the sample, contacting the nucleic acidsample with one or more primers which specifically hybridize to a geneunder conditions such that hybridization and amplification of the gene(if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. Deletions andinsertions can be detected by a change in size of the amplified productcompared to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0185] Alternatively, mutations in a transporter gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0186] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0187] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant transporter gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0188] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1 988); Saleeba et al., Meth.Enzymol. 21 7:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0189] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the transporter gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 79different nucleotide positions, including non-synonymous coding SNPs atpositions 26367, 32860, and 39908. Changes in the amino acid sequencecaused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of of these SNPs that arelocated outside the ORF and in introns may affect gene transcription.Positioning of each SNP in an exon, intron, or outside the ORF canreadily be determined using the DNA position given for each SNP and thestart/stop, exon, and intron genomic coordinates given in FIG. 3.

[0190] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0191] The nucleic acid molecules are thus useful as antisenseconstructs to control transporter gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of transporter protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into transporter protein.

[0192] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of transporter nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired transporter nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the transporterprotein, such as ligand binding.

[0193] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in transporter geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredtransporter protein to treat the individual.

[0194] The invention also encompasses kits for detecting the presence ofa transporter nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in normal nervous tissue,brain, brain neuroblastoma, heart, kidney, lung, spleen, testis, andleukocyte. Specifically, a virtual northern blot shows expression innormal nervous tissue, adult brain, and brain neuroblastoma. Inaddition, PCR-based tissue screening panels indicate expression inbrain, heart, kidney, lung, spleen, testis, and leukocyte. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting transporter nucleic acid in abiological sample; means for determining the amount of transporternucleic acid in the sample; and means for comparing the amount oftransporter nucleic acid in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect transporter proteinmRNA or DNA.

[0195] Nucleic Acid Arrays

[0196] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0197] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0198] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0199] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0200] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0201] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0202] Using such arrays, the present invention provides methods toidentify the expression of the transporter proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe transporter gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 79different nucleotide positions, including non-synonymous coding SNPs atpositions 26367, 32860, and 39908. Changes in the amino acid sequencecaused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of of these SNPs that arelocated outside the ORF and in introns may affect gene transcription.Positioning of each SNP in an exon, intron, or outside the ORF canreadily be determined using the DNA position given for each SNP and thestart/stop, exon, and intron genomic coordinates given in FIG. 3.

[0203] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0204] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0205] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0206] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0207] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified transporter gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0208] Vectors/host cells

[0209] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0210] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0211] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

[0212] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0213] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.Coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0214] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0215] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0216] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al, Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0217] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0218] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0219] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0220] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterotransporter. Typical fusionexpression vectors include pGEX (Smith et al, Gene 67:31-40 (1988)),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology185:60-89 (1990)).

[0221] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al, Nucleic Acids Res. 20:2111-2118 (1992)).

[0222] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSecl (Baldari, et al., EMBO J6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0223] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0224] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0225] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0226] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0227] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0228] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0229] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0230] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0231] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0232] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0233] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such astransporters, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

[0234] Where the peptide is not secreted into the medium, which istypically the case with transporters, the protein can be isolated fromthe host cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0235] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0236] Uses of vectors and host cells

[0237] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga transporter protein or peptide that can be further purified to producedesired amounts of transporter protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0238] Host cells are also useful for conducting cell-based assaysinvolving the transporter protein or transporter protein fragments, suchas those described above as well as other formats known in the art.Thus, a recombinant host cell expressing a native transporter protein isuseful for assaying compounds that stimulate or inhibit transporterprotein function.

[0239] Host cells are also useful for identifying transporter proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutanttransporter protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native transporterprotein.

[0240] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA that is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a transporterprotein and identifying and evaluating modulators of transporter proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

[0241] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the transporter proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0242] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the transporter protein to particularcells.

[0243] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0244] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0245] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G₀ phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0246] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,transporter protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivotransporter protein function, including ligand interaction, the effectof specific mutant transporter proteins on transporter protein functionand ligand interaction, and the effect of chimeric transporter proteins.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or moretransporter protein functions.

[0247] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 5 1 3731 DNA HUMAN 1 tcctgcgtgg agctcccagg cacctgccta agagccagccagcacccagc cactgccaga 60 gagaggcagc tcggacctcg gccccatggt ccaggtggagttctacgtca acgagaacac 120 cttcaaggag cggctcaagc tgttcttcat caaaaaccaaagatcgagcc tgaggatccg 180 gctgttcaac ttctccctga agctgctcac ctgcctgctctacattgtgc gcgtcctgct 240 cgatgacccg gccctgggca tcggatgctg gggctgcccaaagcagaact actccttcaa 300 tgactcgtcc tccgagatca actgggctcc tattctgtgggtggagagaa agatgacact 360 gtgggcgatc caggtcatcg tggccataat aagcttcctggagacgatgc ttctcatcta 420 cctcagctac aaaggcaaca tctgggagca gatcttccgcgtgtccttcg tcctggagat 480 gatcaacact ctgcccttca tcatcacgat cttctggccgccgctgcgga acctgttcat 540 ccccgtcttt ctgaactgct ggctggccaa gcacgcgctggaaaacatga ttaatgactt 600 ccaccgtgcc atcctgcgga cacagtcagc catgttcaaccaggtcctca tcctcttctg 660 caccctgctg tgcctcgttt tcacggggac ctgcggcatccagcacctgg agcgggcggg 720 cgagaacctg tccctcctga cctccttcta cttctgcatcgtcaccttct ccaccgtggg 780 ctacggtgac gtcacgccca agatctggcc atcgcagctgctggtggtca tcatgatctg 840 cgtggccctc gtggtgctcc cactgcagtt cgaggagctcgtctacctct ggatggagcg 900 gcagaagtca gggggcaact acagccgcca ccgtgcgcagacggagaagc acgtggtcct 960 gtgtgtcagc tccctcaaga tcgaccttct catggacttcctgaacgagt tctacgccca 1020 cccccggctc caggactatt acgtggtcat cctgtgccccacggagatgg atgtccaggt 1080 gcgcagagtc ctgcagatcc ctctgtggtc ccagcgggtcatctacctcc agggctctgc 1140 actcaaagac caggacctca tgcgagccaa gatggacaatggggaggcct gcttcatcct 1200 cagcagcagg aacgaggtgg accgcacggc tgcagaccaccagaccatcc tgcgcgcctg 1260 ggccgtgaag gacttcgccc ccaactgccc cctctacgtccagatcctca aacctgaaaa 1320 caagtttcac gtcaagtttg ctgaccacgt ggtgtgtgaggaggagtgca agtacgccat 1380 gctggcgctg aactgcatct gcccggcgac ctccaccctcatcaccctgc tggtgcacac 1440 gtcccgcggc caggagggac aggagtctcc ggagcagtggcagcgcatgt atgggcgctg 1500 ctccggcaac gaggtgtacc acatccgcat gggtgacagcaagttcttcc gcgagtacga 1560 gggcaagagc ttcacctacg cggccttcca cgcccacaagaagtatggcg tgtgcctcat 1620 cgggctgaag cgggaggaca acaagagcat cctgctgaacccggggcccc ggcacatcct 1680 ggccgcctct gacacctgct tctacatcaa catcaccaaggaggagaact cggccttcat 1740 cttcaagcag gaggagaagc ggaagaagag ggccttctcggggcaggggc tgcacgaggg 1800 tccggcccgc ctgcccgtgc acagcatcat cgcctccatggggacagtgg ccatggacct 1860 gcagggcaca gagcaccggc ctacgcagag cggcggtgggggcgggggca gcaagctggc 1920 actgcccacg gagaacggct cgggcagccg gcggcccagcatcgcgcccg tcctggaact 1980 ggccgacagc tcagccctgc tgccctgcga cctgctgagcgaccagtcgg aggatgaggt 2040 gacgccgtcg gacgacgagg ggctctccgt ggtagagtatgtgaagggct accctcccaa 2100 ctcgccctac atcggcagct ccccaaccct gtgccacctcctgcctgtga aagccccctt 2160 ctgctgcctg cggctggaca agggctgcaa gcacaacagctatgaagacg ccaaggccta 2220 cgggttcaag aacaagctga tcatcgtctc ggcagagacggccggcaatg ggctgtacaa 2280 cttcatcgtg ccactgcggg cctactacag atcccgcaaggagctgaacc ccatcgtgct 2340 gctgctggac aacaagcccg accaccactt cctggaagccatctgctgct tccccatggt 2400 ctactacatg gagggctctg tggacaacct ggacagcctgctgcagtgtg gcatcatcta 2460 tgcggacaac ctggtggtgg tggacaagga gagcaccatgagcgccgagg aggactacat 2520 ggcggacgcc aagaccatcg tcaacgtgca gaccatgttccggctcttcc ccagcctcag 2580 catcaccacg gagctcaccc acccttccaa catgcgcttcatgcagttcc gcgccaagga 2640 cagctactct ctggctcttt ccaaactaga aaagagggagcgagagaatg gctccaacct 2700 ggccttcatg ttccgcctgc cgttcgccgc cggccgcgtcttcagcatca gcatgttgga 2760 cacactgctc taccagtcct tcgtgaagga ctacatgatcaccatcaccc ggctgctgct 2820 gggcctggac accacgccgg gctcggggta cctctgtgccatgaaaatca ccgagggcga 2880 cctgtggatc cgcacgtacg gccgcctctt ccagaagctctgctcctcca gcgccgagat 2940 ccccattggc atctaccgga cagagagcca cgtcttctccacctcggagc cccacgacct 3000 cagagcccag tcccagatct cggtgaacgt ggaggactgtgaggacacac gggaagggaa 3060 ggggccctgg ggctcccgcg ctggcaccgg aggcagctcccagggccgcc acacgggcgg 3120 cggtgacccc gcagagcacc cactgctacg gcgcaagagcctgcagtggg cccggaggct 3180 gagccgcaag gcgcccaagc aggcaggccg ggcggcggccgcggagtgga tcagccagca 3240 gcgcctcagc ctgtaccggc gctctgagcg ccaggagctctccgagctgg cgaagaaccg 3300 catgaagcac ctggggctgc ccaccaccgg ctacgaggacgtagcaaatt taacagccag 3360 tgatgtcatg aatcgggtaa acctgggata tttgcaagacgagatgaacg accaccagaa 3420 caccctctcc tacgtgctca tcaaccctcc gcccgacacgaggctggagc ccagggacat 3480 tgtctatctc atccgctccg accccctggc tcacgtggccagcagctccc agagccggaa 3540 gagcagctgc agccacaagc tgtcggcctg caaccccgagactcgcgacg agacacagtt 3600 ctgagccagc cctgcacgga gctcaggcca ccaagcccggggtcctcatg aaggacgtgg 3660 aggagcgtgt gaggacacgg tggcactagc gtgaccctgaggatggcaca ctctacttac 3720 catggatcct g 3731 2 1172 PRT HUMAN 2 Met ValGln Val Glu Phe Tyr Val Asn Glu Asn Thr Phe Lys Glu Arg 1 5 10 15 LeuLys Leu Phe Phe Ile Lys Asn Gln Arg Ser Ser Leu Arg Ile Arg 20 25 30 LeuPhe Asn Phe Ser Leu Lys Leu Leu Thr Cys Leu Leu Tyr Ile Val 35 40 45 ArgVal Leu Leu Asp Asp Pro Ala Leu Gly Ile Gly Cys Trp Gly Cys 50 55 60 ProLys Gln Asn Tyr Ser Phe Asn Asp Ser Ser Ser Glu Ile Asn Trp 65 70 75 80Ala Pro Ile Leu Trp Val Glu Arg Lys Met Thr Leu Trp Ala Ile Gln 85 90 95Val Ile Val Ala Ile Ile Ser Phe Leu Glu Thr Met Leu Leu Ile Tyr 100 105110 Leu Ser Tyr Lys Gly Asn Ile Trp Glu Gln Ile Phe Arg Val Ser Phe 115120 125 Val Leu Glu Met Ile Asn Thr Leu Pro Phe Ile Ile Thr Ile Phe Trp130 135 140 Pro Pro Leu Arg Asn Leu Phe Ile Pro Val Phe Leu Asn Cys TrpLeu 145 150 155 160 Ala Lys His Ala Leu Glu Asn Met Ile Asn Asp Phe HisArg Ala Ile 165 170 175 Leu Arg Thr Gln Ser Ala Met Phe Asn Gln Val LeuIle Leu Phe Cys 180 185 190 Thr Leu Leu Cys Leu Val Phe Thr Gly Thr CysGly Ile Gln His Leu 195 200 205 Glu Arg Ala Gly Glu Asn Leu Ser Leu LeuThr Ser Phe Tyr Phe Cys 210 215 220 Ile Val Thr Phe Ser Thr Val Gly TyrGly Asp Val Thr Pro Lys Ile 225 230 235 240 Trp Pro Ser Gln Leu Leu ValVal Ile Met Ile Cys Val Ala Leu Val 245 250 255 Val Leu Pro Leu Gln PheGlu Glu Leu Val Tyr Leu Trp Met Glu Arg 260 265 270 Gln Lys Ser Gly GlyAsn Tyr Ser Arg His Arg Ala Gln Thr Glu Lys 275 280 285 His Val Val LeuCys Val Ser Ser Leu Lys Ile Asp Leu Leu Met Asp 290 295 300 Phe Leu AsnGlu Phe Tyr Ala His Pro Arg Leu Gln Asp Tyr Tyr Val 305 310 315 320 ValIle Leu Cys Pro Thr Glu Met Asp Val Gln Val Arg Arg Val Leu 325 330 335Gln Ile Pro Leu Trp Ser Gln Arg Val Ile Tyr Leu Gln Gly Ser Ala 340 345350 Leu Lys Asp Gln Asp Leu Met Arg Ala Lys Met Asp Asn Gly Glu Ala 355360 365 Cys Phe Ile Leu Ser Ser Arg Asn Glu Val Asp Arg Thr Ala Ala Asp370 375 380 His Gln Thr Ile Leu Arg Ala Trp Ala Val Lys Asp Phe Ala ProAsn 385 390 395 400 Cys Pro Leu Tyr Val Gln Ile Leu Lys Pro Glu Asn LysPhe His Val 405 410 415 Lys Phe Ala Asp His Val Val Cys Glu Glu Glu CysLys Tyr Ala Met 420 425 430 Leu Ala Leu Asn Cys Ile Cys Pro Ala Thr SerThr Leu Ile Thr Leu 435 440 445 Leu Val His Thr Ser Arg Gly Gln Glu GlyGln Glu Ser Pro Glu Gln 450 455 460 Trp Gln Arg Met Tyr Gly Arg Cys SerGly Asn Glu Val Tyr His Ile 465 470 475 480 Arg Met Gly Asp Ser Lys PhePhe Arg Glu Tyr Glu Gly Lys Ser Phe 485 490 495 Thr Tyr Ala Ala Phe HisAla His Lys Lys Tyr Gly Val Cys Leu Ile 500 505 510 Gly Leu Lys Arg GluAsp Asn Lys Ser Ile Leu Leu Asn Pro Gly Pro 515 520 525 Arg His Ile LeuAla Ala Ser Asp Thr Cys Phe Tyr Ile Asn Ile Thr 530 535 540 Lys Glu GluAsn Ser Ala Phe Ile Phe Lys Gln Glu Glu Lys Arg Lys 545 550 555 560 LysArg Ala Phe Ser Gly Gln Gly Leu His Glu Gly Pro Ala Arg Leu 565 570 575Pro Val His Ser Ile Ile Ala Ser Met Gly Thr Val Ala Met Asp Leu 580 585590 Gln Gly Thr Glu His Arg Pro Thr Gln Ser Gly Gly Gly Gly Gly Gly 595600 605 Ser Lys Leu Ala Leu Pro Thr Glu Asn Gly Ser Gly Ser Arg Arg Pro610 615 620 Ser Ile Ala Pro Val Leu Glu Leu Ala Asp Ser Ser Ala Leu LeuPro 625 630 635 640 Cys Asp Leu Leu Ser Asp Gln Ser Glu Asp Glu Val ThrPro Ser Asp 645 650 655 Asp Glu Gly Leu Ser Val Val Glu Tyr Val Lys GlyTyr Pro Pro Asn 660 665 670 Ser Pro Tyr Ile Gly Ser Ser Pro Thr Leu CysHis Leu Leu Pro Val 675 680 685 Lys Ala Pro Phe Cys Cys Leu Arg Leu AspLys Gly Cys Lys His Asn 690 695 700 Ser Tyr Glu Asp Ala Lys Ala Tyr GlyPhe Lys Asn Lys Leu Ile Ile 705 710 715 720 Val Ser Ala Glu Thr Ala GlyAsn Gly Leu Tyr Asn Phe Ile Val Pro 725 730 735 Leu Arg Ala Tyr Tyr ArgSer Arg Lys Glu Leu Asn Pro Ile Val Leu 740 745 750 Leu Leu Asp Asn LysPro Asp His His Phe Leu Glu Ala Ile Cys Cys 755 760 765 Phe Pro Met ValTyr Tyr Met Glu Gly Ser Val Asp Asn Leu Asp Ser 770 775 780 Leu Leu GlnCys Gly Ile Ile Tyr Ala Asp Asn Leu Val Val Val Asp 785 790 795 800 LysGlu Ser Thr Met Ser Ala Glu Glu Asp Tyr Met Ala Asp Ala Lys 805 810 815Thr Ile Val Asn Val Gln Thr Met Phe Arg Leu Phe Pro Ser Leu Ser 820 825830 Ile Thr Thr Glu Leu Thr His Pro Ser Asn Met Arg Phe Met Gln Phe 835840 845 Arg Ala Lys Asp Ser Tyr Ser Leu Ala Leu Ser Lys Leu Glu Lys Arg850 855 860 Glu Arg Glu Asn Gly Ser Asn Leu Ala Phe Met Phe Arg Leu ProPhe 865 870 875 880 Ala Ala Gly Arg Val Phe Ser Ile Ser Met Leu Asp ThrLeu Leu Tyr 885 890 895 Gln Ser Phe Val Lys Asp Tyr Met Ile Thr Ile ThrArg Leu Leu Leu 900 905 910 Gly Leu Asp Thr Thr Pro Gly Ser Gly Tyr LeuCys Ala Met Lys Ile 915 920 925 Thr Glu Gly Asp Leu Trp Ile Arg Thr TyrGly Arg Leu Phe Gln Lys 930 935 940 Leu Cys Ser Ser Ser Ala Glu Ile ProIle Gly Ile Tyr Arg Thr Glu 945 950 955 960 Ser His Val Phe Ser Thr SerGlu Pro His Asp Leu Arg Ala Gln Ser 965 970 975 Gln Ile Ser Val Asn ValGlu Asp Cys Glu Asp Thr Arg Glu Gly Lys 980 985 990 Gly Pro Trp Gly SerArg Ala Gly Thr Gly Gly Ser Ser Gln Gly Arg 995 1000 1005 His Thr GlyGly Gly Asp Pro Ala Glu His Pro Leu Leu Arg Arg Lys 1010 1015 1020 SerLeu Gln Trp Ala Arg Arg Leu Ser Arg Lys Ala Pro Lys Gln Ala 1025 10301035 1040 Gly Arg Ala Ala Ala Ala Glu Trp Ile Ser Gln Gln Arg Leu SerLeu 1045 1050 1055 Tyr Arg Arg Ser Glu Arg Gln Glu Leu Ser Glu Leu AlaLys Asn Arg 1060 1065 1070 Met Lys His Leu Gly Leu Pro Thr Thr Gly TyrGlu Asp Val Ala Asn 1075 1080 1085 Leu Thr Ala Ser Asp Val Met Asn ArgVal Asn Leu Gly Tyr Leu Gln 1090 1095 1100 Asp Glu Met Asn Asp His GlnAsn Thr Leu Ser Tyr Val Leu Ile Asn 1105 1110 1115 1120 Pro Pro Pro AspThr Arg Leu Glu Pro Arg Asp Ile Val Tyr Leu Ile 1125 1130 1135 Arg SerAsp Pro Leu Ala His Val Ala Ser Ser Ser Gln Ser Arg Lys 1140 1145 1150Ser Ser Cys Ser His Lys Leu Ser Ala Cys Asn Pro Glu Thr Arg Asp 11551160 1165 Glu Thr Gln Phe 1170 3 48667 DNA HUMAN misc_feature(1)...(48667) n = A,T,C or G 3 agaggcacct gtggcaagcc ctggggtcccccaaggagaa cggagcggag gggagaacaa 60 cagagctcgg ggaggccgtg ctgagaggcccagggagtag aggagattct ggggtctggt 120 caccgtgaag ggcatggtga ggctccctgccggagaggag agggtgggag gcagtaggag 180 caagtgaggt gagcagaccc cccgcgttgaggcttttgcc atggagggcg ggacagtggt 240 agggaggcgg ggagggtggg ttcctaagctccatgctttt agggcccggt ggtccctggg 300 taccgccggc agtgagggga ggagggccttaggagaccct gggctcagcc cccgggggca 360 gctcccgcct cagccctgcc tgcccccctgacatggccag accaggccca gcgcagcctg 420 gacctccagc atcctgtctg agcgaaagacatccattcag agagaaagac ggcggacaga 480 ccccgcaaag agacacttat gcagggaggggcctccccag ccggggctcc aggcacacag 540 aggagaggcg tggggcagga ggaggggctgagggacacag cagagctggc tacgctgagg 600 ggttgcaggg catgatgggg cactctgggggcagactgac cccccattag cactggttca 660 gacgccactg gtctgtggga ggcttgtcccactgcctggc cccaggagcc ctgagtatgc 720 ccggtggggt gacaagcctg cccccaggacttggcctgag cctcctcttc agcatggggc 780 aagggcaccc agagccacac gcctcttcctggaccctgga cccttcttca tttcatttat 840 ttatttatta ttcatttttg agacagggcctcgctctgtc acctaggcta gggtgcagtg 900 gcgtgttgac ggctcacttc agcctcagactcctaagttc aaacgattct cctgcctcag 960 cctccccaga agctgggact acaggcacgtgccgccatgc ctggctactt tttattgaga 1020 cgggggtctt gctatgttgt ctaggctggtctcaaactcc cggcctcaag cgatcctccc 1080 acctctgcct cccaagttcc tgggattatgggcacgagcc actgtgaccg gcccctggcc 1140 ccttcttgag ctctgtaacc agcagaggccactggggcag atcaaggcag gtgctgggac 1200 tctaccaagg cccctcccgc ccgccccacagcagccccac cacgaaggct gctgcacaca 1260 cctggtccac accacttacc ctgagtgttgtgagctttgg gaaacatcga atgttttaat 1320 cactttcgga gatgttgggg tcaccagccccaggccaggc agtgacatca aattctcaga 1380 ttcgattgat aggagccagt gcctgctcagaggggaggtg aggctcacag cccacccact 1440 gggtgtcaca gcctcctgcc ctggtggacagtgacaagga aggggttgag gcaggaacag 1500 gggttaggct ggtgcttacc tcccccgacttctggacagc tgccctggga cctctgcttg 1560 aacactgcct cctccacgca gccgtccaggatccttccag gcagcacaag tcttcctcca 1620 ggctccgctg tctgtctgcc ctactgtgttgtggccctcg cgctgcactg ggcagggggc 1680 ccatgctgag gagctcactg gactgcgtgttctgccccca caccatccct gagggaggcg 1740 gggcacctct ctttgacccc agggagcaaggccaggcacc ggaggcagcg cccgagggct 1800 aaggcagccg actgatagga gcagacgtcatggagggcac agcggccatg gctgggagca 1860 gccgagggtc ccatggagga cagcccagcaccccagggca cgggccgttt agggggacat 1920 gtgctccccc ttcccagaga cggccatgcctgctgcagga accctggctc tgggtcttgc 1980 tcctgtctgt cctgcaggct ggcagcattggagttttcct ctcaggggat cattaaaccc 2040 tggcctagcc tggcccgcat cccagcacagccgggcacag gagaccctgc gtccaaggct 2100 ggggcagtgg gggcagcagc cgtccacccccaggctccat cctactcttg gtggccagcc 2160 tgggtcatgc aggtggccac ggtaggtgagccacgcaatc tccccacccc acctcacccc 2220 acctgccacc cccggcaact ggcacactgtggtctcttct caggaccgcc ggggcccggt 2280 tggaggccag agggctgggt ggtaggccccatgagcagcg gcgtgcatgg gggttgaggg 2340 gtgtctggga accagtggct ggggccataggtgagcctga gggaagggga gcggcactgg 2400 ctggaggggc cccacagctg gcctgcgaggtgggtgcagg gatgaccact tcccaccagc 2460 ccatcttccc tgacagccct tgaggctgcagtggcccata gccttggagc acagccacca 2520 gcggcctcca gacagagcag ctgaccatcagctgtggaca gcctgtatga cgcaggtcct 2580 acggcatgtt ctgagcactt gcggtgtcctgagcaccatc cccagggcca tatgggcaac 2640 tttacctcct ggcaggtgcc ctgcacccacggaggcttgg gcgagggtgt gtggagtgag 2700 tgccgcaccc tgagccccaa cagggcggcctggagggccc agactcacag cagcgagtgc 2760 ccagggctgc ggggaggaat cttcagaggagccgaggctg ggtcggctgt ctgctgggtg 2820 tgacggaccc caggaccctc ctgggtctgaggtggaggga gtggtcctgc tgggccctga 2880 cttctcaact cctgcagttg gaaagttggaagaagtcagt caggttgggg ctcccggcgt 2940 gtccccagcc tgagtcccca ctggccctgagcctccatgc ccctctctgc ttcttcaggg 3000 tccaggtgga gttctacgtc aacgagaacaccttcaagga gcggctcaag ctgttcttca 3060 tcaaaaacca aagatcgagt gagtggggtgcctggagggc cactcccagg cagggaggtg 3120 ttgagggcgc cgggagcaag cctggcgatggcgaaggctg gggctgtgag ctcagggaga 3180 gtccatgggg tgggagccac ctgagtgctgcgggggcatt tggaagcagg gtgggggtgg 3240 gtctgcatgg tgagtggggc acagggtagccccctcttag cttcacactg tctgagacca 3300 gggcctctgc agcgggactc gtggggacaccgtcctgcct gctggacacc aggctgggtg 3360 gggcaggacc atccataggt gccagcaaggtctcccctgt ggccacgccc tgcctcccac 3420 ccctccatac agccccaccc tccccaacagctccctgccc cgttcccagc cagagcttgg 3480 catcatgctc cccccgggat tgcaggtgaggacttggggg gtttccccag gggctccaca 3540 ttcacctgcc tgctccccaa tccctatcctgtcctccaag aagtggagtg gcctctagaa 3600 atcccctgca ccccaagttc aggcacctgtaaggtggggc cagggacaga gcctcttgga 3660 ggctgaccca agagcatcag cccccaaacaacgcagggca cacagagctc tgcgtgcagc 3720 ccgggtgtgg gaggggagcc cagctggggccatccagaga gcccagccag acccgggtgc 3780 aggccctgct ccccgaagcc cagagctgggtcagagccct gaggccgctg ccctccccgc 3840 aggcctgagg atccggctgt tcaacttctccctgaagctg ctcacctgcc tgctctacat 3900 tgtgcgcgtc ctgctcgatg acccggccctgggcatcgga tggtgggcca cgtgcgcggc 3960 cgggcgcggg gtcccgggtc ccagggctgagccttcccac tgggccgtta cagaagctga 4020 tggcgtccct gggggagccc ctgtgcctggggccttgggg agtccttcct tccccagtgc 4080 ctgtcccagg atggaaaaag ccaaggactcagtgcccagg ctcccgcaga cggctcagcg 4140 tagggagaag ggtccccatc gtagacttccagctgtgccc ttagctacta gcggacccag 4200 aaggtccacg gaaaggctgg agcatctaggatgtccctcc ctgcccccac aagtgtctct 4260 gtgctcactt gagcttctgc aggtcagggaagggggcgcc cagtccccct ggggaaagcc 4320 cctacctgtt tctgggggtg tcttctacccctcctgggtc ccaaggagtc ccctccccag 4380 ggcccatcgt cctggggccc atgctccagggacttctggg agaggtgctg ggggcagcaa 4440 tctgcaccgt caggtcctta ccagcctcgcagcagccgcg atgagggtgg gtgggaactt 4500 gctctctctg agtcctgttc tgtctccttctgctgttgct gcttcgagaa gtggcggctg 4560 ctgggaccta ctctgtgccc agcgtgccttggcaactcct gctcacctct gtgtgggtcc 4620 caggggacat cagcagggcc ctgcggccctcggtgccggc ctgtacctga gtcatgtctt 4680 tggggaccag cccccaaccc caggcctctctctctggctc cctggcactc ggatgctgtg 4740 ctctgtgtcc tgtttcttct ctcccctctcctgcctcgtt cccttgttca ctcgtccact 4800 tgttctgtcc tgcctcctgg tttccagaaaatcctttctc tgaatgtcct tccgaatagg 4860 tgtctccagt cctttcctgt tttacgctttccccatctgc tgggcctgtt tctcccgagc 4920 tcacattggg ccctgacttt ttcatacccgtagatttcct caaatgtctg gtgccgttta 4980 tgcgtaagag tgctcaggcg ctgaggggctctgcagccac gtgtgtggtg tggttgtcac 5040 catccagcca tcggggtgaa ccctgccagcatgctggtcc cccctctggc tgcgcagagc 5100 aggttcttcc ctggagagaa ggcctgactgccgggagcct ggcttctggg gacctaaagg 5160 cgtccatgta aacttttgct caatcccccttttcacctgt taccccgtca caagggggcc 5220 tggcatcccc aggccagaga ctttccctctcctaagaacg aacctcctgt ctttgtctag 5280 gtcagaagag ggcagaaccc actaggagagtttgagaagg agaactgggc cctgactgac 5340 tctcaagcta ctttcctagg tcactccccaccccaagtcc cagggtcccc tggggcttct 5400 ggtttcacat ccccacaggg cccaagtctgttgtgttcac tgcccgtccc ctagcacagc 5460 gtccaggaca tggatggggg tgggaagatgggtggatgat ggatggatgg ttggatggat 5520 ggtggatgat ggatggatgg acggacggacggatggacgg atggatggag tggatggagg 5580 gatgagtgga tgggtggata atggatggataggtggatgc atggtgggta gatggatgga 5640 tggatggaat agatggatgg agggatgagtggatgggtgg atgatggatg gatggatgga 5700 tggatgatgc gtggatgggt ggagggatggatgttggatg gagggatgag tgaatgggtg 5760 gagggatgag tggatggatg gagagatgagcagaaggatg gagggatgag tggatggatg 5820 atggatggat ggatggacag ataagtagatggatagatgg atgagtggat ggatgatgag 5880 cagatggttg gagggatgag tggatggatggatggaggga tggatggatg gacagatgag 5940 tagatagatg gatgagtgga tggatagagggatggatgga tgagtggatg gatggaggaa 6000 tgagtggatg gatggagaga tggatgatggagaagtggat ggatggatga gtgagtggaa 6060 agatggatgg gtggatggat gatagatggatgtccagata cttctgtggg cacttagttt 6120 gcaacaaccc cattaaaaat caggggaaagtggatcagtt gaaagaaaag acaagaggag 6180 ctggccatca ccaggggcct tgtgagcagtgaggtctcct tggtcagtga ccctggcatc 6240 gatgatggtc tgggacaaaa ctagcttgttggccatgccc tggtcactgt cgcagcagct 6300 ggttccaggt ggcttggggg ccgcttgtcctcagaagcag cttagggacg tgcctcttgg 6360 agtcagcacc tggggatagg agcttcctctccctgtctct tgaacactgt ccagccttgt 6420 tcaaggcagc tctggccttg gggaggccctgagtgttccc acatgctctc tctggccatg 6480 taaggcccct ccctgggagc ctgttgtcagcacaggcaga accagtgtgt tgctgttggg 6540 ttggggctac ccccaatccc gcagatgtgggatgtaccct cgctgtctgc ctgctgggct 6600 gagcagtggc cagcccagca ttcctcagttagaggccctc ctgcaggatc tccgcagtag 6660 gtggggaggg ggcaggctgc cttgtctcccaggtgttaga gctctgatgt gggggttagc 6720 cccgggtggg tgagtaggag gcccccatgaacccgagcct gtggaagccc tcgggcagca 6780 agtccttgca gtggtgctag aggggccagggccgggccag cgctgtgtct ctccacagct 6840 ggggctgccc aaagcagaac tactccttcaatgactcgtc ctccgagatc aactggtgag 6900 tccacactcc agctcccaat agccaggcgctcagaggcct gggaccaggg tggggtggga 6960 tgagtctcca ctccagctcc caatagccaggcgctcagag gcctgggacc agggtggggt 7020 gggatgagtc tccactccag ctcccagtagccaggcgctc agaggcctgg gaccagggtg 7080 gggtgggatg gacagaggct cacactgcctggtccctgag ggataatgcc cagcctcaac 7140 ccagcagcca cccaggttcc ccaggacacatgtgccctta gccctggctg tggtcaatgc 7200 tgggaatgtc cccttcaaga ggagcagtttgagaattgtg agttcccagc cagttccaca 7260 gcctccacac gctcttcctg agcccagtgagagatgcaag gaagcccgtc tcccagtgag 7320 ttggcggctt ctcttccagt tcaccatcttacggcaaagg aaggcacctg ccccgggcag 7380 gcagtgggag gtgactgcgg ggcaactggggtgcctggtc cagagattaa gacctgtccc 7440 cagaacctgc aaagcatgtc cccagtgcctgagcccctgt ccgcatcagg caaggagcag 7500 atggccccct gcagccatgc ccctgcctgtttatggatga ggaactgaga ctcggggaaa 7560 cccagggggc tgtgggttag atagcccgacctccctgctc ttagggacat tcatgggagc 7620 ccggcttcag ggacccaggt gaaaaaaacaggctacccag tctggggccc aggctgctgt 7680 tgcctgtcgt gtccagagag gagaagctttgccacccttg gtagtgtcat gggctgcagg 7740 ccaaggctcc atggagactc agccatgggccctggcctca caagcacctc ctggggggcc 7800 tgcctgctgc agtcctgctc catcctcctctcttggggaa ccttcccttc cctcctcctt 7860 atggtcccct ccaaaggcaa ggtaggctcctttggtccag ccccaatgag cagcacatgc 7920 ttggggccag tggaggccaa tggttatggccccagcccca gggtggctgg gggacactag 7980 tggctgtggt gccctactgt gctgcctcctttctcttccc agggctccta ttctgtgggt 8040 ggagagaaag atgacactgt gggcgatccaggtgagtgcc ctaccctgcc cccctcccga 8100 ctgcagtggt gctcagtaag cactgaggaccaacccagac tcagtaagta gggaacccag 8160 gctcggtgag cactgaggac atacccagtctcagtaaatg ctaagaacaa acccaggctc 8220 agtgagcact gaggacagac ccaggctcagtaagcagggg acccaggctc agcaaatgct 8280 gaggacaaac ccaggctcag taaacaggtgactccaactc agtgagcact gaggacaaac 8340 ccagcattta ctgacccagg ctcagtaaatgctgaggaca aacccaggat cagtaaacag 8400 ggacccaggc tcagtgagca ctgagtacagacccaggctt agtaagtagg ggacccaggc 8460 tcggtgagca ctggggacaa actcaggctcagtaggcagg ggacccaggc tcagtgagca 8520 ctgaggacag acccaggctc agtaagcagggaaaccaggc tcagtaaatg ctgaggacaa 8580 acccaggctc agtaaatagg gaaaccaggcccagtgaatg ctgaggacaa accaagactc 8640 agtaaacagg tgactcaggg tcagtgagcacttaggacag acccagcatt tactgactca 8700 ggctcagtaa atgctgagga caaacccaggctcagtgagc actgaggaca gtcccaggct 8760 cagtaagcat gggacccagg ctcagtgaatgccaaggaca aagccaggct cagtaaaaca 8820 ggtgactcag gctcagtggg cactgaggacaaacccagca tttactgacc cagggtcagt 8880 aaatgctgag gacaaaccca ggatcagtaaacaggggacc caggctcagt gagcactgag 8940 gacagaccca ggctcagtaa gcagggaaaccagactttag tgaatgctga agacagacag 9000 gctcagtaag tagggaaccc aggctcagtgaatgctgaag acaaactcag gctcagtaag 9060 caggggtccc aggttcagtg agtgctgaggaccgacccag gctctactgc tctccacaca 9120 attttcccag ggatccctga ggacttcctgaccccctctg tctgcttcac cacctgaatc 9180 tctccctcca ctgacccatc tgtgtccttgctgttttcct ggggtggaga gtgcacaggc 9240 caggtccctg gctggctcct gggtgggtgacctagttcat gactgggtcc catgtctcca 9300 tctgagacca gagagggtct ttcacacccacccctatcat gttccagagg ggtcagtaga 9360 tgcctgtgct gagaggggtt ccgtgggggaaaaccaggtc cactgagcct ccatctccgt 9420 tccctcccca ccagggcagc gggatagccgctctctggtg tcttcctgag cgtcctgcct 9480 cacccactgc accctccatc ccaccctgggcctagagtgg gcggcccagg aggaccatgg 9540 tttcaagact ccatctgccc tcggccctggcgggggtcag ccaaaggcct tggtggctaa 9600 gactgtcctg acatgggagt gagggtcaaggcagacccca gacacacacc tggctttgtg 9660 tggtacctgc ccgcgaggcc tgtggggtcaggccccagcc ccagccccgg cctgctccag 9720 agctccttcc ctttcctgac tcccaggtcatcgtggccat aataagcttc ctggagacga 9780 tgcttctcat ctacctcagc tacaaagtgagtgcctgccc gggatggcac ctcacagggg 9840 gtccccaccc tccccagcct cccnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9900 nnnnnnnnnn nnnnnnnnnn nnncccacctcccccaccct ctcctatttc cccacacttc 9960 ccagcctcat ccactgacct ccagggtgggctcctcgccc tcttccccac cctgcccctc 10020 ccctgctggg ccccaccccc accctccatcgcccccgctg ataccccccg tttggcccca 10080 gggcaacatc tgggagcaga tcttccgcgtgtccttcgtc ctggagatga tcaacactct 10140 gcccttcatc atcacggtgg gtgagccccagctgccagga gtgcgggccc tggagcccca 10200 gccctgacct gtccccttca cagatcttctggccgccgct gcggaacctg ttcatccccg 10260 tctttctgaa ctgctggctg gccaagcacgcgctggaaaa catgattgta agccggggcg 10320 gggggtgcag ctgggacttg ggggggccaccctcagcctc accggccctg gaagacactg 10380 tgcgacgtag cctgccacgc cccggccctggcatgacctg tagaggaccg ggaagccagg 10440 gcagcagaaa ctctgtctct gctcctggaaaacatccaag ccaagccccg aatgccctcc 10500 ttccaggaag aaactcaatc ccagagcttttcctaagcct ggaaatgaaa tggctggcag 10560 gaggaggagg gacaaagtga agggtgtgacaaacccaggc cacgaggacc agggccacaa 10620 acaccaggcc acacgcaaac tggggccacggggccacagg caggccctgt gctgagcatc 10680 tcaggaaacc tgggccacgg ggccacaggcaggccctgct ctgagtgtct caacatggtc 10740 ccctgtcatg tgaatggcag cagtcagttcccttaggcgc ccaccctctc caagccagac 10800 cccttgactc acagccccca ttcctgccccagtggtaaat cagcgttacc atgctgggtc 10860 caagccttgc acactaacca gccctctgacctgagactta agcctcatcc ccggggatgt 10920 aggcaagatg gggacccgtc ctgagagaggacagcagagg gggtgcccta cccaggtgca 10980 ggctgcccgt gggcctcagc ggagacgcaggtgtggccag gcgtgcaggc tgccccgtgg 11040 gcctcagccg atagcaggtg tggccaggtgcaggctgccc catgggcctc agccgagaca 11100 caggtgtggc caggtacagg ctgccccgtgggcctcagcc gagacgcagg tgtggccagg 11160 tgcaggctgc cccatgggcc tcagccgagacacaggtgtg gccaggtgca ggctgccccg 11220 tgggcctcag ccgagacgca ggtgtggccgggccgtctgc tttccactgt ccttctagtc 11280 tctattcatt ggctcctggc ggggtccacagtccctgccc gctgacagcc accactcctt 11340 ccacagaatg acttccaccg tgccatcctgcggacacagt cagccatgtt caaccaggtc 11400 ctcatcctct tctgcaccct gctgtgcctcgttttcacgg ggtgagtgcc ggccgtcagt 11460 gtgagcaccc caggacgttg ggagggcccgagaggcaagc agggccgggc gaggggatac 11520 agatgcctat gtccaagcta tcggggcagaaaaggccaca gtgcctgggc tgcgggtgtc 11580 gggccaccaa gctgggactg aggtcaggaggcagctccaa gcccacgtcc ccagtacacg 11640 agcagccctg cagcccgact cctccaaggacagagatacc cagatctggc ttcctggtct 11700 atgccatgga cgtagagaag gggactggcccctaggccag gtggggtctc ttggctgagg 11760 cccagctgaa agcagggtct ggaggcagccagggtaaagg tgggggtgcc cagagctgcg 11820 agggcctcca gcccacccag gcatgcccactgtgcccacc tgcctgtgtc ctcgtggagg 11880 gctccatgtt gctgctctgc cttgggtcccagcgaggcct ggtcaccact tcccgtcccc 11940 aggcagggat gtcaggcaag cactgtgccctgggggaggg agagtgccct gcgtttcccg 12000 cctcccttcc cccctgcccc tcatgacagactgacagaca cagagctgag tgggcagatt 12060 ggggcatcca tgaggatagc atctgggacctggcggcgac cccagccctg cccattagac 12120 ctcccagcct caggcctggg cgcttgtctggctgtgccgg gcagaggcct gagtgtggtg 12180 ggtaaagggg caaggctctg agatgggggtagagggccag accccaggcc cacccctgtg 12240 tcacccaagc ccacgctgat gacacagccctgcatcccct gctcccagag aatgttccag 12300 ggacctagga gagagccacc cggcaggcagggaggctccg gggaattcgc cgtgaacaga 12360 ggccgccatg ctgtggccaa gctgcattgtcagccagcgt caggcaggag gtggctccgg 12420 cagagcttgg ggacagatgg gcagggctgagggcctgatg ccacccagct gtcaggaggg 12480 cggggctcgc ctggtgatgc acagctcagtctcctgggca gtgagggtcc cgtgggcagg 12540 caggatctct gaggggccac ggccccccagctcctgggcc ccaggccgcc cctcactgcc 12600 aggggttgca ggacctgcgg catccagcacctggagcggg cgggcgagaa cctgtccctc 12660 ctgacctcct tctacttctg catcgtcaccttctccaccg tgggctacgg tgacgtcacg 12720 cccaagatct ggccatcgca gctgctggtggtcatcatga tctgcgtggc cctcgtggtg 12780 ctcccactgc aggtgggtcc tctgggcaccagccctgggt ggcaccagca aagggacagg 12840 cgggtgccag tagagggagg gtgccactgaggctgtggca cagtgcgggg gccactccca 12900 ggaggggaca gtgaggccag gcgggtggtgcctgctccgt tgcacgcccc cactgagggt 12960 ctacggcggg tccggtggtg ctcagcatggtgggtaatga tggagtcccg tgagctggcc 13020 tcttccttct ggggagatgg tgggtctccagtgccagggt gacctgcccc tcccaggccc 13080 aggcagagtg cagggaaggg tcaaggtggacagccggccc atttcccatc cacagccagg 13140 tgcagcagca gctgccaggc ccacagggggcacacccccc cggccacccc agtggcttcc 13200 ccgtcaccac tgctgtggcc cactgcccactgagcagagg aggggacggg gcaagacctc 13260 agtgggaaag gtggaggcct ggagagggcagctgcctcag ggtgtgaagt gcttgggcct 13320 ggactgcctc cgacacctcc tccaggcaccccagcccacc ctggagggac cctgctattg 13380 gggagatggg agaaggaggg gacccctgtgggtggtggaa cattttccag gaggctgggt 13440 aggaggaaga gcctgaggag gtggccagggccttctggga gacagacccc caggtggctg 13500 caggatgccg gggagacagg gcagtgctcctagggagcct ctgctgaccc caggctcagc 13560 cccagctccc tcccgctaaa cagcagtgggcgtggcccag gtatgggccc aggccaggcc 13620 tggctcttct cctcactata tccaagccaaagctgtggca ccagctgtac ggcccccagc 13680 gtgggccatg ttctccacat ctgtggcttctgttctcctg agttcagatg gggccgtgcg 13740 cgtctttcca tctggttgtc ggccactccaccgtccagtg ccacgagtcc cgctcctgtg 13800 agctgcccgc tcctctttgg tcttccccctggttctcgct gatgagcgag tctctgtgtt 13860 ctagaagaag ccagttgtgt gtggctttcccgttacttcc tgctctgtga ccgcccctct 13920 cactttgctt acagaatctt ccattcacggatggtcttga tttttttttt ttaaattaga 13980 gatagggtct cactctcatt ttgttgcccaggctggcctc gaactcctgg gctcaagcga 14040 tcctcccgcc tcagccccag aaatagctgggattacaggc ggcttggctt tgattcacca 14100 atctttccct tatggtttct gttttcctgtctttactgaa aagatcattc cctccctcga 14160 tcataaacct ggcttcctat tttcttctaaaagtgtaaag ctcgcctctc acctggaggg 14220 tttcacccac caggaaccac catgtggggtggggtggagc tgggttctcc cctctgacgc 14280 ctgctccccc acaccctgca ttctgctcctttctctgcgc atttcataac caggcaaggg 14340 cgggagtgag ggtgggagtg aggccaggagcacagtgtgg ggggcactcc gtgcagtgac 14400 actgcctccc tcctcggccc tcctgccccaatccgtaaat ctcacctcaa attctttacc 14460 ttaattactt ctgcaaagac ccttttgcaaatgaggactc atcctgaggt gcagggggac 14520 ctgctttcag ggccatcctt gaccttgctacgggccccca gcctccgtcc ctggtccgcc 14580 ccggcccaca cttcaccacg tggccggcacctgctgaggc cgctgcaccc agatgctctg 14640 cagaggcgtt gacccatcca gtctctgaattccccacaag cccttccaga gaaacatccg 14700 agaggcccct ggcccagccg aggtcagaggaagggtcaac agggccaatg cgtccccctt 14760 tcccctgtgg gcccacggcc cagccacacgcagctccacc ttcgggctgc gtccggctcc 14820 tctccccacc ccacacaccc cagagcgaagaggagcccca gccccagcca cccccagggt 14880 cgctttcaaa taaagcagga gcgaagcgctctctcccgtg cttctcacga gaccgtggca 14940 cccacgggta agggccaaat cgggatgtgcagcaggcctg ttccatgtcc ctctgctgcg 15000 tccacagcct ccgggccggg gctgccttcccattctgctc ctgaagcacc aggggagccc 15060 ccacctcccc ttatctccat taagaagtaagacaaggcca ggcgcagggc tcatgcctgt 15120 aaccccagta ctttgaaagg ccaaggcgggaggatcgctt gagcccagga atttgagatc 15180 agcttgagca acgtagagag actcccatctctaccaaaaa gtatgaaaat tagctgggtg 15240 tggtggcatg cacctgtagt cccagctacttgggaggctg aggcaggaga atcgcctgag 15300 ccctgaggtt gaggctgtgg tgagctgtgatctcgccatc gcactctagc ctatgcaaca 15360 gagagtgaga ccctgtttca aaaagaaaaagacaaaaggg ccaggcacaa tgggtcctgc 15420 ctgtaatccc agcactttgg agggccgaggtgggtagatc acttgagatc cggcgttcaa 15480 gaccagcctg ggaaacatgg caaaagcccgtctctaccaa aaaataggaa aaattagcca 15540 ggcatgatgg cacacagctg tagtcccagctcctcgggag gctgaggtgg gaggatcact 15600 taagcccagg aggcagaggt tacagtgagccaagatcaca ccactgcact ccagcctggg 15660 tgacagagcg agatgccatc tcaacaccatcttaaaacaa acaagacaag gtgactccag 15720 gtgcaccagg tcctgcaggc atcacacctgcactgctctt cagggaaatt cacggaacaa 15780 atgcgccatc gtcacgagaa ctgattgcatcgatgggtat ggccatggtg acaagtgacc 15840 gacagcccag cagcctgcac gcagaggtgtcactgtagct cgagctccgt gtccacagtg 15900 gggtccctca gatcagccac tgcctgactgccttgtctcc tttctcccgt gtggggcagg 15960 cagagcaggc cctgagtggg acaagctctgagcgaggcag ggaggaaggc gggggcaggg 16020 cctgggggct ggcgcaccct ccctcccatcagccctgccg gacctggtgc tcaaacctga 16080 cccaggtggg cctgtgctgt ccctggcagggctgggggtg attgggacag tgatgccgct 16140 gaccccaccg tctaagccgc tccctgcctcctccgaatga ctggaaagac tgagcctctc 16200 actggcgatg gtaagggcgg gctgcagtgcccttgccctt ggtctccact gggctgaatg 16260 caccagtaaa agcccttgaa gcagagtgatgaccccgagg ccctcgctca cgagcgtcct 16320 tctggaacac agcagcgccg gctgggtctcctggaagtat cctccaggct cttcgatcag 16380 caagacagac aggcagctta aacagcagatatgtatcctc cgccagccct ggaggacaga 16440 atcggaagtg gaggggttgg cagggctgaccctatcaggc cgctttgggg gagattgtcc 16500 catgcctctc tccagcttct ggtggggccgcaatccctgg ggctgcttct acggtggctg 16560 tagaaacctc agcctctgcc ccgtgtgcacttggcctctt ctctgtacct ctcactggat 16620 ttagggccca ctccaaccca gggtgaccttatcttaaatc attacatctg cagagaccta 16680 tttccaatca gatcacatgg cgaggttctgggcagatagg acttttaggg tcactacgga 16740 gctgccgtca ttagctcctt catcctcacggcggccctgg agggttgccg tgccatgcag 16800 ccatcttgta ggtgaggaag ctgaggctcagagtggggga cgtgagccct ggggtgtctg 16860 acagcagagc ccccgccagc accatccgcggggaagcctc tgttccggtc gccctggagt 16920 cttgagcacc tcccagctgt ggaaactgttttgtgtggca aggcagtgga gcctcgtcac 16980 cggaacaaag caggcgctgg ataagtgcaatgcgatggtc ccatttagac cagacgatag 17040 cacatcagcc atgcagccca cagtccatgggagctcctgc cattgttcat ttaacacgtg 17100 ttcaacaagc cgggcaccat gactcagcctgtaatcccag cacttcagga cgacaaggca 17160 ggaggatcgc ttgagcccag gagttcgagaccagcctggg caatgtagtg agaccccatc 17220 tctacaaaaa ataaaaataa aactagctggtcatggtggt gatgcaccta gaatcccagc 17280 tactcaggag gttgaggcag gaggatggcttgagcccggg aagtcaaggc tgcagtgagc 17340 tgtgattgca ccacggcact ccagcctgggtgacagagtg agcctccgtc tcaaaaaaaa 17400 gaaaagccaa agactttcac ctagaggccagtggggccgg tgctgggagg ccccggtggc 17460 ctctgttttg gatctgtccc ctttgagtctcaacacccag agcttgcatc tggggagcat 17520 gagccagggc aggcagcatc ctcagaccagcagcctcaaa ccacctgccc cccagacata 17580 ggctcctgac agtgaggcct gcgagtgcctggaagaccgg ggtctgaggc ctgatttggc 17640 cacgcttacc atcaagagag tgatccccaatcccacgggc tgagcctccc caccctcggt 17700 ttactccaca gtctccagcc gcccctcctcccttggtccc cacacatcct gccagccggg 17760 cctgcgtcct gctctgtgtc atctgaggctatcagcatct acagtttcca tgtgggaaag 17820 gcccccctaa tgtaggtgtc accccccggccggccctgcc ccaggcccct aacagacagt 17880 cgtgtgtcag ggcccaggtt cttcaccgggagccctcgct ccccaggcct ggtcgctggt 17940 gctcacctgt ttctcacctg cagttcgaggagctcgtcta cctctggatg gagcggcaga 18000 agtcaggggg caactacagc cgccaccgtgcgcagacgga gaagcacgtg gtcctgtgtg 18060 tcagctccct caagatcgac cttctcatggacttcctgaa cgagttctac gcccaccccc 18120 ggctccaggt gaggcccctt accgtggcccagcagacgac tccctcccgg cccctagaga 18180 cgccatcctc tccccaggac tgcttggcaagtccctgagt cctctcagcc ttggtttacc 18240 ccttcagtga gggcagatgc ctccctcccggcgcccagag acgccatcct ctccccagga 18300 ctgccatcct ctccccagga ctgcttggcaagtccctgag tcctctcagc ctcagtttac 18360 cccttcagtg agggtggcag cccacagtcaggtggagggg ccctgcgggg gcccctcttt 18420 tccctcccac cagatgcaag atcacagtgagctctagggc ccagaggtgg tgggcacaaa 18480 cacagtcctg aaggcagggc cgcccgggcggggcaggggc tggctcagag ggtctgaccc 18540 tccgcctggc cggcaggact attacgtggtcatcctgtgc cccacggaga tggatgtcca 18600 ggtgcgcaga gtcctgcaga tccctctgtggtcccagcgg gtcatctacc tccagggctc 18660 tgcactcaaa gaccaggacc tcatgcgagccaagtgagtg ctggtgggcg gagggggtgg 18720 catgggggca cctttcctga gtcaggtcggctgctcaggg ctgaggcatt gaccgtcgct 18780 ctcctggcca atgagagcca tgagagcattatagactatc cccagggtgg accatctagg 18840 tggactgtcc agggtggatc gtccggggtggaccatttag ggtggatcgt ctggggtgga 18900 ccattcaggg tggaccttct gggatggaccatttaggtgg accatacagg gtggatcatc 18960 tggagtgaac tgtcaagggt ggaccatccagggtggacca tctggggtgg accatctagg 19020 atggactgtc tggggtggac cattcagggtagaccttctg ggatggacca tttagtgtgg 19080 accatccagg gtggattgtc tagggtgggtcatctggggt gaactgtcca ggggggacca 19140 tccagggtgg gtcatctggg atggactgttcagggtggac catccagtgt agatcatgtg 19200 gggtggacca tctagggtag actgtccagggtggaccatc tggggtgggc tgtctggggt 19260 gggccattca gagtgaacca tctgggatggatctctaggg tggaccatcc agggtggatc 19320 atctggggtg gaccattcaa ggtggaccatctagggtgga ccatctgggg ttggtcctct 19380 ggggtagatc atcaggggcg gaccgtctggggtataccct ctggggttgt ctggggtggg 19440 ccatccatgg tggaccctct tgggtggaccatctggggtg gatcgtctgg ggtggaccat 19500 ccatggtgga ccctcttggg tggaccgtctggggtggatc atccagggtg gactgtctgg 19560 ggtggaccct ctggggtgga ttgtctggggtagactgtcc ggggcctctt gcagtttctc 19620 tagctcaggt cccaggccca gatgagcaggaccctgcagc caggcccttg tccctgcacc 19680 atacccactg tggagcagcc caggcaagcccaacccagct aagtctctgt ctcccttttc 19740 agggtgaggg gcagcctttt gcgagctccaccttcccctg ctccaagtct agggaaatct 19800 ttgtcaagtt ctagggaggc tcaaaatagaacaggggcgg gagagtcagc tgctgtgagt 19860 caaggtggga gaacgggctg aaggtagagttccttttaaa gacaaattga cagagtgaca 19920 aggtaacaag agtatattat tacctggaannnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnccccccccccg ccaacccaca tcacagtgtc 20040 ccccacgggc atccttgtgt gccctccaccagcaggcatc cctgtgcccc cccccccggt 20100 atcccagtgc cccccccccc cccgcaggcatcctgggcct ctccagactc cctggcccat 20160 ccctggcagg ctctgagccg ggtcaggactcctggacacc ctaggagact ccaagctccg 20220 acactcgaga aggcacagga gccacacctgtcccccgagc gtgtgcaggc tgacagaggg 20280 tgggggcagg gttcaggagc cacacctgtcccctgagcgt gtgcaggctg acagcgggtg 20340 ggggcagggg tcaggagcca cacctgtcccccgagtgtgt gcaggctgac agcgggtggg 20400 ggcagggggt cagatgctat ggatggtgtgtcctggagac accagctgac agcttctcct 20460 ggccacggcc ctgagtggga gcccatggtgcttcctaggg cagtgcttgc ccttcctccc 20520 ccaggccctg ttatctgaag caaccccccagcttgtccct aagttggcca ggtaggaggg 20580 gcatcaggag gtcgtgcgag ggggctctgctggctggggc tgcctggatt tgtccatact 20640 cccaggcgtc aggggctagg ggtccagggttggcaggagc aggcccggtg gagaccccac 20700 ctaggtgtgt gccagtgacc tgggcactggcttccggggt acccatcttg tatgttgcca 20760 tgagacattc acccagggct gggttggggtgcaggaggga ggggggaatc tgggggcaga 20820 gaccctgtcc gctccctggc ctggctactctgtggtctgg gaacctgggt ggtcacttcc 20880 agcgcctcct gggtctcctg aaagcccagctgcagaaacc acctagcact gctgagacgc 20940 tgtctaagaa actgtcgcct ctgccccagggcagggaacc ccaccacatc actggcacca 21000 ctgtctgctc cgcaggggcc ccaggccctgtacccacagc cctgggctca tccctgcctg 21060 gctgggaatg gcctacgccc ttgccctctccccactccct gggcccaagc aggtgcatcg 21120 cctgtgtgtg ctggggacgg gaccacaaaagaggtggcgt tccataggca atgggttcca 21180 gcttggggct ctggacatgg cagggggagggggagtgatg aggagagacc ctgggagtac 21240 caccaacact atgccactaa ggacacattcaggctcctgg aatgaaaatg tcaccaggag 21300 ggttttaaat tccaggtaat aatatactcttgttaccttg tctctctgtc aatgtgtctt 21360 taaagtaaac tctaccttca gcccgttctcccaccttgac tcacagcagc tggcattccc 21420 gcccctgttc tattctaagt ctccctcgagacatactaat acaaccccaa aaaactagca 21480 gggaaaggtg gagctcgcaa atttttaaaactcctcctga ggcttgtagg aattcactaa 21540 ttggcatgtc attcggctat gtgcacacctatgggtgggc attattttgt atgagaactc 21600 ctcatttcct cgctcatggg tcaatattagagacaaactt attagtgatc aaggaattac 21660 tgtataaaat ctagaatcat ttgcctatatttaagagacc agacagtcca ccctggatga 21720 tccaccccaa aaagtccacc caagagggtcgtccatggaa cagggacccc tgccggtgca 21780 gggcagataa ggatgcccgt gggggacactgtgatgtggg ttggcggggg ggggggggca 21840 gtatggggat gcccactgag ggggcactgtgactcctgac cagcagagag taggggcctc 21900 ctcccgcctt ccatcctccc cgccttccatcctctccgcc ttccatccag ccgtcctctc 21960 agtctctttc tgtgcacctg ctgcaccagcctcctcccag aggaggtcct ccccacctca 22020 cctccgcacc cccggctgca ctgcccacctcccctgctcc acccacgctc aggccctggt 22080 gcattgcagg atggacaatg gggaggcctgcttcatcctc agcagcagga acgaggtgga 22140 ccgcacggct gcagtgagtg aggctgaggccctgcccagg caggaggggc accgtggggc 22200 cggggagcgg gggtccctga gggaagagacctgccccagg ctgccggtgc cgcccagcag 22260 cccacagagg ccagcccgtc tgcactgaccaaccacccac cccgccagga ccaccagacc 22320 atcctgcgcg cctgggccgt gaaggacttcgcccccaact gccccctcta cgtccagatc 22380 ctcaaacctg aaaacaagtt tcacgtcaagtttgctggtg cgtctggggc acacgtgggt 22440 gatggtgtat ctggggcagg gcacgtgtgcacacgtgggt gatggtgcat ctggggcagg 22500 gcgcatgtgc acatgtgtga cggtgcgtctggggcaggac gcgtgtgcac acgtgggtga 22560 cggtgcgtct ggggcaggat gcgtgtgcacacgtgggtgt cggtgcgtct ggggcagggc 22620 gcatgtgcac acgtgggtga cggtgcgtctggggcagggc gcgtgtgcac acgtgggtga 22680 cagtgcatct ggggcagggc acgtgtgcacgtgtgtgtcg gtgtgtctgg ggcagggcgc 22740 gtgtgcatgc tgttgggtga tggtgcgtcctggggcaggg cgcgtgtgca cacgtgggtg 22800 acggtgtgtc tggggcaggg cgcatgtgcacgcagttagg cgagctgtgt ggggcagcgg 22860 gaggggctgg cccctggagc tcctcacacaagcacaccag gaggtgctgg aggggacggc 22920 agacccccat cctcaccgca tccgagaagggacctagggg gtccaaactc ttcagatgaa 22980 gtcttatgct gggatcctgg ggtcagtgaaggcagggtca gaggtcaggt gggggcagga 23040 gcaccgtctg atgagcacct ctatgggcagggaccatgcc gggtgcccgg ggaacggggg 23100 gcaggcccca tgccaggtgc ccaggggacaggggtcaggg cccacatgga gtgcccaagg 23160 cacaggggcc agggcctgcc ccgccctggaactcctcgct gagctgggag agaagcacag 23220 gagcgatgga aggtccaccg aggctcagaccaagtagggg gttgaggtcc acagactctc 23280 ggggcagaga tgctgaagcc ggacagcagacacgggggtg ccaggcaagg gtgcatctgc 23340 atagcacctt caggaagtga gcaggtactgtgggggagga gagagccggc agagcggcag 23400 gtggaccggc ctcccccact gcccgcagaccacgtggtgt gtgaggagga gtgcaagtac 23460 gccatgctgg cgctgaactg catctgcccggcgacctcca ccctcatcac cctgctggtg 23520 cacacgtccc gcggccagtg agtgccccgtgccccggggg accgacctcc atggcggggc 23580 cggcgcaggg agacaacgca gggcctgcttgggggcgggg atgggcttcc cagaggaggg 23640 gcacatggcg ggcaaaagtc ctgcatagggaggggatcca tgccagggga agcagagggg 23700 ggcacctgca gacccagccg gggagaaggggcagccatgg ccgagggtga cgctcccctg 23760 gccccgccct ggcccacagg gagggacaggagtctccgga gcagtggcag cgcatgtatg 23820 ggcgctgctc cggcaacgag gtgtaccacatccgcatggg tgacagcaag ttcttccgcg 23880 agtacgaggg caagagcttc acctacgcggccttccacgc ccacaagaag taaggccggg 23940 ctgcatccac agggctggcg ctccagggctgctctgctct gtgccctccc caccctcccg 24000 gtcaggcaca ggggtggccc tggggcggggctgcagaggg ctcgggggag ggcatcaggt 24060 catcctgcct ggcgagggca gccgcaggactgggctccgg gtccacataa aaacctgcca 24120 cgcggctcct ccctgaggct tgtgggctgaccccagctct ctggtcccca acacctctgg 24180 gacgggaggg ctcagccaag gtccctgaccccaaatggcc cccaggagga agacgcggag 24240 ctccggtggg gactctggtg atttgcaggaagggcaggca gggagcggga cagggcaggt 24300 gagcggcggt acctgaagtt gccggtgcctctgcccaggt atggcgtgtg cctcatcggg 24360 ctgaagcggg aggacaacaa gagcatcctgctgaacccgg ggccccggca catcctggcc 24420 gcctctgaca cctgcttcta catcaacatcaccaaggagg agaactcggc cttcatcttc 24480 aagcaggagg agaagcggaa gaagagggccttctcggggc aggggctgca cgagggtccg 24540 gcccgcctgc ccgtgcacag catcatcgcctccatgggtg agccgggaca ggcgcgcggg 24600 actccctggg cctgctcctt tggcgggagaccaggcggga caccggcagg tgaccaggtg 24660 ggatgggaga ccaggcagga cagggggaggtaaccaggtg ggacaggaga ccaggcagga 24720 caggggcagg tgaccaggtg ggacgggagaccaggcaggg cgggagacca ggtgggacag 24780 gagaccagac caggcagggc aggagaccaggccaatgtgg cccccagacc cagttcctct 24840 tggcctatgc ctccaacctt ggacaggtggagtctctctg ggcctgtttt tgaatctgtc 24900 aaacaggtgt ccccgccttc ctgtggccacctttgtgggc attgctcact gtgtccaagt 24960 gcctgtccaa gtggggccgc ccacaggaccggttggcagc tcaaccggag gctcctggtg 25020 gtcccatcag cccggagggt ctgcttcacgtgtgtccctc aaacagtcgg gagtcctgac 25080 gtccactggg gccaggagtt ggcggaatgaggtcgcagtg gccgagggct ttggcctctt 25140 ctcgtgcctt cagggatcct cccggggagccgcttaccag acagggtcag gctgcctctg 25200 agcaggtgga ggcccatggc ccctagggcaccacccatag tggtgttgcc ccctccccag 25260 ccaggccttc ctgggggaca gttagggagcagggccaggc caggaagcca ctcaggccac 25320 agacccacag ccgggccagc atgttgccacctccgtacag tggcccaggc agaggctgac 25380 cctatggggc accccagcac gcccaccctggggtgttgtt acacggtggc ttctggggca 25440 ccaggtggtt cagtgcagtg gggcacagtctccaagaccc agagggccct ggggtttgga 25500 gacgtgccga tgcggagccc accacctgccagaccccgca ggttcccggc cgccgtctac 25560 ggcagctcac tcggggggcc gggcctggccgcagggcact ggggaggcag gctgctgctg 25620 cctagccaca cctccacctt cacctgcgcagtaggcactc tgcccccagg tggcttccag 25680 gaaaccaggg tgactcgggc aaccctcactgtacccacag aggccctggg ccatacctag 25740 aggtccacaa cccccatctg gcagcctggggtggtgcagg agtggcagag tctgctccca 25800 caggcactga gtcacccagc tgctccccacctggccccat ccaccaggca gcctgaccag 25860 ctgcacaggc ccctaaggct gagacccccgagcccagaat cagccaaccc cctcctcagg 25920 catctggtgc tgaggccaca gcagctggcctgggtggcac cgatggggca ctggggcccc 25980 ctgggtcttc gctcaacatc atcgccaccccagaggccac tgtccctgtt gtatggaggg 26040 ggaaactgag gcatagattg aagctcctcagctggagcac aggagccagg ccatgacagg 26100 gtctccagag ctgactgtgt tcacctgagctctgggaact cgccgcccat ggagggtggg 26160 agtcgggctg tggccaagca cagggctctcttccagggac agtggccatg gacctgcagg 26220 gcacagagca ccggcctacg cagagcggcggtgggggcgg gggcagcaag ctggcactgc 26280 ccacggagaa cggctcgggc agccggcggcccagcatcgc gcccgtcctg gaactggccg 26340 acagctcagc cctgctgccc tgcgacctgctgagcgacca gtcggaggat gaggtgacgc 26400 cgtcggacga cgaggggctc tccgtggtagagtgagtgct gccttggaga cggctcccag 26460 tggggggagg agccgcccat gagtgcgggggatgggtgtc ggagcatcct tggtggtccc 26520 ccatgctctg aattgcagct tctggacagctccgtggaag tccttgtttg acaaatgaaa 26580 tcttcagggg gcccaacaca gacatcagggccacatcacc ctcgtcgccg gggagcactt 26640 ttgagtgtca ctgagatggg gtgtgctgggctacaccctt tccagttggg gctgggggtg 26700 cagagcttat tggagcgagg cagctcactggcacaggggc tgtcaggcac caggcagcgc 26760 caccctcaga gaggggcccg ttcccacccacctcaccagc cccatagtgg cccggccact 26820 ctctgaatca ggatggagct gggcagtgaccccaggccat cctccaccca gtgccctagg 26880 tctccccctt agcactacag agcacagaggggcacagccc gcctgaccag gggtgggtgt 26940 cctctgagca gggtccctgt gcaacccctgggcaaggcag tgtcccagcc tggaaaccac 27000 agcccgtcct gagttgttgt cctggcctaggtttggggcc aagtcctcct gcttcaaatc 27060 atgagacagg aggccccact ctgttctcagcagcttctgc cttttggcct cagccaggac 27120 agacaagtgc cccagctgca gggcccgaggtccatcctca gcggggctgc ctcactctgt 27180 cctggccttt gcctctgggg tggggtcaacacactgtaat gacagcccag ctgctgggaa 27240 acagccctga accattgcgt gtgtgtgttgggtgctgctg gggacggggt ggtggcctgc 27300 tggggacaac agcctggctg gacaagtatgtgggtaagag cccaaggcca gagtgcctgc 27360 cccaccagcc gggggtgctg gggatgtcagggaggcatgg cgggcgggca gagccctgtg 27420 ggttttgcat gtggctgaaa agcctggtctaggctgtggt gggaggagaa agaccgagta 27480 gggcatgggg gtgggtgtgc aaggggggtgtgtccggtgt gtgtgtgtgg tgtatgttat 27540 gtgtgtggtg tgtgtccata tgtgtaatgtgtgttgtgtg tcaacgtgtg tttgtcacgt 27600 gtggtgtgtg ttgtgtgata tgtgatgtgtgtctgggtgt gtgtggtatg tgtccgtgtg 27660 tgtgatgtgt gtctgagtgg tattgtgtgtggtatgtggt gtctgtgtgt tgtgtgtggt 27720 gtgtgtccgt gtgtggtgtg tgtggtgtctgttgtgtgtg tggtgtgttg tgtgtggtgt 27780 gtgtgtctgt gtgctgtgtg ttgtgtgtctgggtgtgttg tgtgtccgtg tgtggtgcgt 27840 gttgtgtctg tgtgtggtgt gtgttgcgtatgttgtgtgt ccgtgtgtgt gtggtgtgtg 27900 tctgtgtgtt gtgtggtgtg tctgtggtgtgtctgtgtgt tgtgtgtccg tatgtggtac 27960 gtgttgtgtc tgtgtggtgt gtgttgcatatgttgtgtnn nnnnnnnnnn nnnnnnnnnn 28020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnngt gttgtgtgtt gtgtatgttg 28080 tgtgtcgatg tgtgtgttgt gtgtctgggtgtgtggtgtg tatgttatgt gtccgtgcac 28140 gtggtgcacg tgtgcatacg cctgcatgtgttcatttctg tgcgcatgtg ttgtgtgtgt 28200 gtccgggtgt gtccatttcc gtgtgcctgtatggggccat gtgttatgcc gagggctgac 28260 catggggggg tcctgggctg accaggtgaggggtgagccc cacgccaccc cctcccacac 28320 ctgggtgagg gcagcagcag tgagggccctacccacccct cggtctcctc cagtccccca 28380 aggccctgat ggggctaagt ccatgcctccccagccctgg ccttgcagcc gctgctgacc 28440 cgccaggcag gcacgggtcc ttaaggttcggcacctgcat gggtgccctg ggcactgccc 28500 tgactgccct gcaggacaca ctgggtcctgggtgccaagg ctaggcaggc cctatgacac 28560 ggtgaccaca ggctcagctg gaggagcgtttgctgatgca caggtggtta agggaagaac 28620 gggccacctt gggtcagctg tgcgtgaccccgagcaacgt ggctgtgggt ggagtgggtg 28680 ccctcgagtc ccccagcccg aggggggccccagagcagac ccagccacct ctgtgcagct 28740 gtgctgaggg ctcctgtctc ctgccccaggtatgtgaagg gctaccctcc caactcgccc 28800 tacatcggca gctccccaac cctgtgccacctcctgcctg tgaaagcccc cttctgctgc 28860 ctgcggctgg acaaggtaag gctggcggctctgccgcgct ctgcaccccc agacgccagc 28920 accgggccgt gcatacctgc cctggtttctctttggtcac tttacatttc gataccattg 28980 caaacttcta gcacagctgc aagaattctgcaagggagag ccacagatcc ttcacccaga 29040 ttcagcagac ggtcccttcc tgtcccgtttgggctctccc tctcacagtg tctatgttag 29100 aggcacctgt tttctgagcc atcttgagaacaggttggag ccaccacacc cctttcccct 29160 aatacatcta catgcgttcc caaagatcaagaaccatctc gtaactacag ctcattagga 29220 cagggacccc ggcccggcat gggtgtctgatccacagtcc aagttcagat tgggccgagg 29280 gccccgaagg ccttcagcgt cctgcgtggcagtgttttcc ccaccgggag cctgtcccgg 29340 ctgcgtctta gcaactcctt tctcgttactctcccttaat ctggaatgtt ccctggggtc 29400 tcttgacctt aatatttttg cagagcagggaccagtgacc tggtggcagg tgggtctgcg 29460 atgtttcttg gtggtgggac tctgcctgcatgtggggcca gccctccctg gaaactcggg 29520 ggtcaattgt caggtcactg gcgatgtttacttggatctc atggcaagct ggggtcagta 29580 gggtttcccc actgtgaaaa tgactgttttccttctggag ttagcagctt ctctgtgggg 29640 gacgtgttga gatcaggaaa gcaaaccactcgacccagcg gcttcagcat ccctcagtgg 29700 gttctttttt gtttatttgt tgtttggttttttttaagac ggggtcccac tctgtcatcc 29760 aggctggagt gcagtggtgg gatcatagctcactacagcc tccacctccc aggctcaagg 29820 ggtcctccca cctcagcctc ccaagtggctgggaacacag gcacgcacca ccacacctgg 29880 ctaattttat ttatttattt attttttgtagtgatggggt ctcactatgt tgcccaggct 29940 agtctcgaac tcctggactc aagtgatcctcccacctcgg cctcccaaag tgctggggtt 30000 acaggcgtga gccaccatgc ctgccatctgtggatggttt ccagttccgg aatctctctc 30060 cagctgtgtg ttagggctcg gccacggaggggacctcccc ttccccatgt gtgtgtgtcc 30120 ttcacggacc acaggctccc agcatattctgtgggttata atctgtggcc ggtcattgca 30180 gtagcatcat ccttgacaga tcgttattgattctgatgct cggaccccct cagttcactg 30240 ggtggggtgg gggccttcaa gcggcttctgggtgcttctg acacgatccc ctcacccttt 30300 gagctcatct gtgctttcca accggagatgttccagccac atcttggcct tcgcctgctc 30360 cagtctggaa tgaaccactt ctccaaggaaccctgattcc ttttagtgga aaatgatgat 30420 tggaaaccaa gacctgggca cgtggcgtgctcactgctgt tggcatccct gctctgagaa 30480 aacgtgtgtg catacacatg acacgtatctatgcccacac ctagatctct agccacatgc 30540 caaaaccaca tgttcacatg gacaccgccctttccacccc tgacatgact gctctgtgcc 30600 gcctgtgggt acagctgtgg catctggtggtgcctgtagc tccccacagg cgtgtgcagg 30660 ccgtgaaagg gctgtgggcc gagaggctaacggctgggtg ttcacggggg atgtttggtg 30720 aggggtctgg gagggctcca gtggccagcaggaaccacag ccctgactcc agccctgact 30780 ccagcatctg cccccagggc tgcaagcacaacagctatga agacgccaag gcctacgggt 30840 tcaagaacaa gctgatcatc gtctcggcagagacagccgg caatgggctg tacaacttca 30900 tcgtgccact gcgggcctac tacagatcccgcaaggagct gaaccccatc gtgctgctgc 30960 tggacaacaa gtgaggctcc tggggctcagcccaccccgc ccacccgggc cctcagacct 31020 gcagccagca gcctccccaa ctgggcccacccttcgcctt tgcagagggc acgggaacat 31080 ggggcctctg gcctggtcct ctcagctttcctaaaaaggg ggactctcct tcctgctccc 31140 aactcctcct gctcccagct cctccccacacccactcctg ctcccagctc ctccccaccc 31200 ccagctcctc tggctcctgg gtcctccctgctccctgttc ccccagttcc cagctccacc 31260 caactcccag ctcctccttg cttccagctcccccagttcc cagctactct ccattcccag 31320 ctcctcccca ctcccagctt ctccccactcccagttcccc ctcactccca gctcctccct 31380 gctcccagtt cctctggcca ccagctcctccccactccca gttcctctgt ctcccagctc 31440 ctccatgctc tcagctcctg tggctcccagctcctcccac tctggtcctc tctccccctt 31500 tccccctcct cccttgtcac tccttctgtcctgtttgcct cctgctccac tcactctgag 31560 ccccaggatt ggggtggagg gataaatggctcttcctcct gggcaccttt ttgcccaggg 31620 gaccctagga ccctgacagc tgagcccagggtcatcttgg ctgtgtgacc tcagcaggtc 31680 ccaccctccc gggccttggt ttccccttgaataaaataaa gaatggccca ctggccttaa 31740 agtactcccc aggtcccata cgctgcggttctggggaacc cctgcctggc ccagctctgt 31800 gcatggaggg tagggcccca ctgggcctgaggagggcagg ccttgaagca gggtgggccc 31860 ctccaggacc gctgtcccca caggcccgaccaccacttcc tggaagccat ctgctgcttc 31920 cccatggtct actacatgga gggctctgtggacaagtaag gcgtggccgg ccgaggctcg 31980 tgggggctcc acacccaccc ctcccctcctcttccaaagt ctggggtgac cccgaccgca 32040 ggtggggtgg ggggctgagg tcctcctgccttctgaccaa atcccggggt cctgtgggtg 32100 gggagtgggc cgcatcctca gccacgggccctcggtcccg ccaccagcct ggacagcctg 32160 ctgcagtgtg gcatcatcta tgcggacaacctggtggtgg tggacaagga gagcaccatg 32220 agcgccgagg aggactacat ggcggacgccaagaccatcg tcaacgtgca gaccatgttc 32280 cggtgcgtcc agtgtccggg gctcggctctaaaccacccc acagccacga ccacgggccc 32340 tcgccctgag acccccacag ccacgaccacgggccctcgc cctgagaccc ccacagccac 32400 gaccacgggc cctcgccctg agaccgccacagccacgacc acgggccctc gccctgagac 32460 ccccacagct atgaccacaa gcccccaccctgaaaccccc acaaccacag ccatgggacc 32520 acaccctgag acccccacag ccatggactctgcccagtag acccccacag ccacgaccac 32580 aagccctcca ccctgagagt cccacagccatgaccatggg cctctgccct gagacccccc 32640 acagccgtgg gaccctgccc tgagacccccatagccaaga cagtgggccc tgccctgaga 32700 cccccccaca gccatgggac cccgccttgagacccccaca gccacatgat catgggcccc 32760 caccctgaga cctcctacaa ccaccatgggccccgccctg agccgcctgc ctcccccagg 32820 ctcttcccca gcctcagcat caccacggagctcacccacc cttccaacat gcgcttcatg 32880 cagttccgcg ccaaggacag ctactctctggctctttcca aactagaaaa ggtgagcagc 32940 cctgccccgt gccagctgcc accccagaatcccagaaaga gtgggagaaa ggggctcagg 33000 ggaaaggggg ccagtgccat gggaggctgggctcctgccg ccctcctgct ggggaactca 33060 ggagatggcg tggggggccc agcatggacagggtgctctt gatggtggaa ccaggagatg 33120 gaggcagggc gtttccctga ccgcgtgtgaggcactggga atgtggccca tagcagcctt 33180 ccatctccct gagcagggac caggcctggccgtatctgga ggccaaggcc atctgtcctg 33240 gcatggtcag ttggtcagaa ctcccgtggggagccctcag atcggtggtt ccacataggt 33300 tggccagtag ctcttagtac agataacgcacacggctgca ccattccaga cttctccgct 33360 gccctggcca ctacgcccga ccttgaaacaggttcagtat aaccactgcc tccttgtatt 33420 gacaagggac ggagggcctg agagggaaggggcctgcccc ggatcgcaca gtggagcaag 33480 tggctgagct ggaattggct ttctgcccttggagctccat gaagggcacc acccagggtc 33540 aggtgtggaa cccaggggca cgggcactgttgctgttgcc ttgatatctg gctgacccca 33600 gccccaccgc attccccacc tgctcaagctgggacagcac caccttgtcc acacgggggc 33660 tggtgcccag gcagccctag gctgagagcagggagcaggc accttggtca gcagcaatgc 33720 tgtctgctct ccacgaaccc cgagctcaccggtctgggct gaagtcctca tcggcgagct 33780 gctgggacct gcgtggctgc aggattgtgacaccgtggag gatctactga gccaggcccg 33840 gcctggccac caacaccagg cagaggacacacacgggcac tccctgcgac acggacatgg 33900 ctttgtccca cctgaacccg atggagagtgaaagtgtgtt ccggccagcc gtggaccaag 33960 agcccatagg gatcctggag tcagttctgccccagagaag cgagaggagg ccgggctggg 34020 cagccaccag gtcaacaggg gcttctgccttagagatgcc agcctgaggc gtagggggat 34080 gctcgggcac ctggtctcag cacaggagagggctctgtct gccctgcccg ccgcccaagc 34140 ccaccctgga gcctgccctg cccagcgtcgggagctcctg gccccagtcc ccggctcagc 34200 cggcagaggg gtgtgctctg cagctggaaccacggaaggg gaggaagttc acaggattct 34260 gtgttcgggt gccgggccgc tccccagggctgggaggact ctccggatct ggaagaccaa 34320 gtctgaccct gtgtggacag ggatgctgccgtggagtcgg ggtcgaggca gtcatcctgg 34380 cgcggctcct ccggcctcag ccttgcacctctctgtccca caaggtggct tgaagtttgg 34440 gtcagcaagc acgcagccaa cacgctcctgcccgccttcc cgccaggcag ccatcgccaa 34500 tcacccacgc tccgccccgc cgtggggcccatctctttcc ccggcttcac ctccactggg 34560 gctctgtgtt gccggggcgg gtgcggccagctctccatct cagagcagtg aacagtcctg 34620 ggctccagct ccagtcatgc gattccgtggcagttgtgtg acagggacca aggagaaatc 34680 aggacatcgg caaagcctct ggaagcagccgtgagctcct ctgggctgac atggtgctgg 34740 ggtccatgtg ggacagatgc cctgggccctgggcagggcc aggcatatac agagacgtgg 34800 agccgaggga ggagagggtg ccgtggaccacctgacccat caccctctgc agaggctggg 34860 atccgcctcc ccagcatgca aggagcctgtagaaggcacc aggggctgcg tcttctccag 34920 cctgggtggc cttggcccca gatcccccatgggtctctct gacgggccag catacggtga 34980 cctgcttcca acaaggacac ctgtggcagcttcagatcct tatttaaaag acattaatgc 35040 caatttatta taaaaatgta catgtttagtatgtatttta cttcaaagaa tactgaaagt 35100 aaaaaaaaaa aaaaaattga agaaaagttaccccaaatcc ctgcagccaa catcttctac 35160 atccaggccc agtggaagga taaagacaggattccaggca cgactccgga aggaaaaagg 35220 gaggaggtgg agatggcata cacgaccgtcagtgagatgc tctgccaggt gcagcggctc 35280 atgcctgtaa tcccagcact tttagaggctgaagcaggaa gatcgcttga ggccagaagt 35340 tcgagaccag cccaggcaac atagcaagtccccatctata caaaaaattt aatggctggg 35400 ggcggtggct catgcctgta atcccagcactttgggaggc taaggcggac ggatcacctg 35460 aggtcaggag ttcgagacca ggctggccaacatggagaaa cctctctact aaagacacaa 35520 aaattagccg ggcgtggtgg ctgtaaccccagctacttgg gaggctaggg caggagaatt 35580 gtttgaaccc gggatgcaga ggttgcagtgagccgagatt gtgccactgc actccaggct 35640 gggcgacaga gcgagaccct gtctcaaaaaaaaccaagca aaaaaatgaa actattatta 35700 aggatgctgc tggtggggtg acatgcatctccccttgagg gctggctcca gaaggctctc 35760 agagccccgg tgtgtgagag gagtctctcccctgaggccc acctcccctg ccgtccacct 35820 cccctggtct ccagcaccca ggactcctccaccacttcat tgctttcaca tggtcagcct 35880 gtgtgatctc tcctcctcac caggagccttccttcaggct ggctctgccc atcctggggc 35940 tgcacggtgc ctgtcctgct ccctctgaccgggggtctcg tgctcaggaa gggctcatcc 36000 gctcctgtgt gctcctgtct tcactgtcacttgtgaatgg cacccgctca gcccctgctg 36060 aagccctcct gtccagaaag caggaggaaatctctggaag gtcctgaaac gtccagcttt 36120 ttctttcctt ccacgtctct ctctggccctgtcgtggttc ttgtttcctg tttagcgttg 36180 ttctgagtaa aggaaaatta aaattgcaagttgttctgga atttcacgtt cgtctccgcg 36240 aagtacggct ggaattctgg atgcaaatcaccacgattgc aaactcacac gcccatcgca 36300 gctcatcgca cctggttctt gccccaagagcggaaacgcc gcacgaaact aattccactg 36360 tttctcacgc cccagccttc ccaccggccactgatgagta agggcgaacc cggaggaaaa 36420 ggggcttctg cctgcttgtc ctgtcgctgtcgtgtctttt acctgtgaat cgagttctgg 36480 ttccgggggg aagtgtggcc tccgtgggcgcacacgcctg cctgtgcaca cttgcttgga 36540 ggccgagctc cagggttggg gccctccgctgaagaatgac ccgagagttg gccacggccg 36600 ggtccggagc ccgtggatgt accgcctcacgtgacagaag ggcctctgca gatgggatta 36660 agtcacgggc ctcgagatgg gagatcctcctggagcatcc ggcaggttca cgccgtcagg 36720 gtccttctag ggagaggcca gaggtccccatccggtggga agaagctgca cggccagctc 36780 agaagaggga agagggagcc acaagccaaggcaggtggcc tccaggagct ggaaaaggca 36840 agagaaggag gcccccaggc ctccagagggagcggggccc tgcccaggcc tggatccagc 36900 ctgagacacc tcaaactccc gactccagaaacgggagaga ggagacaggt gtcgttttag 36960 ctgctaagtt tgcggtcatt tgttacgggagccccaggaa actcagtggg tccattgcaa 37020 gcctggagca cagggacggg gaggccaaggacagcaggga gcccgatgct tgctcctcct 37080 cagcccagac tccacacgcc ctccggcaccacttacaaaa caccggggga ggagaggatt 37140 atcaggagga tttggaattt cgggatggcaagagcagggc gcttgaccaa gcgcagtgcc 37200 catggcagcc gccgggagtc caccttctcatcagagcccc acatcactcc tcccgggctc 37260 tggccctgag tgtgtctggg aagatagagctggttcaggc ctgccggtgt atgcacacat 37320 gcagacacac actgtgtaca cgtggacacacacaccatgc acaaatgtgc acacaggtat 37380 catgcacaca tgtacggtgc acacacagtgccctcgaccc cgtggctgcc gtgccactgg 37440 agggacctcg gcaccagccc atctgaggcccctcctttcc cacagaggga gcgagagaat 37500 ggctccaacc tggccttcat gttccgcctgccgttcgccg ccggccgcgt cttcagcatc 37560 agcatgttgg acacactgct ctaccaggtcagcggggaag cggcagcagg agggtggcgc 37620 ctgggtggga cccccgtcat gccctcagctcttcagcctg gtccctgttc tgaatgatag 37680 gactccctct gaatgacctt ccctggattccaaggagacc tctgggccct gtccttgccc 37740 cagggatttc tgggcccttt ttgacctatgggtgcctggc aagggagttt tcttagaaaa 37800 ggcctcccag aactctgcct gtgggtcatgtggtgcttgg ggacctggtg gttctgtgtg 37860 tgtgtatgca tgaggggtgg cgagcccgtggccggtgggg tatggacctg tgtcccacgc 37920 ccgtgcccgc gtgcctcact gtggctccctcccttcnnnn nnnnnnnnnn nnnnnnnnnn 37980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnncctc cctccctccc tccctccctg 38040 gccagtcctt cgtgaaggac tacatgatcaccatcacccg gctgctgctg ggcctggaca 38100 ccacgccggg ctcggggtac ctctgtgccgtaagtgcccc tggctgcgct gggctggggg 38160 cgtgctgggc tgtccaagtg ggtggatgggcacctgcccc tgattcacgg tggccaggag 38220 gctctgggtg ctgccacctg ccccagccaactcagggttc ccaccctgca gatgaaaatc 38280 accgagggcg acctgtggat ccgcacgtacggccgcctct tccagaagct ctgctcctcc 38340 agcgccgaga tccccattgg catctaccggacagagagcc acgtcttctc cacctcggag 38400 gttctggggc agcctggggg ctgggactgtggcagcccct gtcctgtgtg acccacagca 38460 tccccacctt ccgggggctg ggactgtggcagcccctgtc ctgtgtgacc cacagcatcc 38520 ccaccttcag tgtcagggac ctgggctagatcagctttgc tattgctggc agctccttcc 38580 gtctggtccg tgtgcccacg tgtagcgcttccagctggag cagccatgac ccctaccggg 38640 ggcagagagg ctcaggggag tcttcaggaagaatacgggc agcccctgtg ctgcagtcca 38700 cacagcagca ggcaccgtgc ccaccagccctaagcatgtt ccgtgcagac cccaggctga 38760 ggcggcgtgg ggggcagggg tgcgcccacaggtcccagac tgcgcctgtt tcctttgcag 38820 ccccacgacc tcagagccca ggtaagcaacccctccgtgc ccacgcagct tctgcggagc 38880 accagaacat cgcagcttca catggagaagcctgccaggc cccacccacc cacccaccca 38940 cagggccctg ggaaagccga ggcaggagcgggtcccacgg atgtgtcggc cccagcacgg 39000 ctcagagaag gctgttgaca ctcactccctcctgcactgg tggtggggaa gctgaggcct 39060 ggggggagta gcctgtgagt ttggacaccaggtgcacccc cgccccctcc caggccccac 39120 caaggcaggc agcccccatg cccatctggcctgaccagcc cccagcctcc caggcctttc 39180 tgaaggtgcc acccaggtgg gatgcctacctcttcccagc accccacgac ccacaggctc 39240 tggcacccca cgttccccag gttcggggcctccaaagggt gtgtttgcaa gagggatcag 39300 ggctcatctg cagatggaga aattgacacgcagagggggt gacctctcag ggcaaggcca 39360 ggatgtgctg gggtccagat gggtcccaggccggacccac agaaataagg gctcagagag 39420 gcgaagggag ctgccctcag cagcctggcgaatcccttga ccaggcccgg gcagggtggg 39480 tggcccggcg atctgtgctc aggaggaaaccaggcacatt ctttccagag gaggaaggca 39540 catgcaccct ggggtctgtc gtcgtcctcttccctaacac ccccagctgc tcccacgcag 39600 gccccaagct gcagagccca cacctcttcctaagcccctg ccccaaagcc cgcgaccctc 39660 ccggcagcct cacccctccc cgccctgccctgccctgccc tgcccagtcc cagatctcgg 39720 tgaacgtgga ggactgtgag gacacacgggaagtgaaggg gccctggggc tcccgcgctg 39780 gcaccggagg cagctcccag ggccgccacacgggcggcgg tgaccccgca gagcacccac 39840 tgctacggcg caagagcctg cagtgggcccggaggctgag ccgcaaggcg cccaagcagg 39900 caggccgggc ggcggccgcg gagtggatcagccagcagcg cctcagcctg taccggcgct 39960 ctgagcgcca ggagctctcc gagctggtgaagaaccgcat gaagcacctg gggctgccca 40020 ccaccggcta cggtaagggc acacggcgcgggtgggggcc ggacggacgg actgggccag 40080 ggtcgggcca cagccaagaa cagtcggccacggcgtcccg gggcccgggc cgtcctcctg 40140 tctgtcatct gtctgtctgt cagcctttctggccaccctc tgcctgcctc tactgtgggg 40200 ccagctgtgg gcaccaccac ggcccagcagaggccccgtg cagaggaggg aagtggccat 40260 ggcccaccct ggtgctggga ggccacaggacccttgggca tccctggtgt gagccccgta 40320 gggaggtgct cgcttccact gctctggggcctcagtttcc ctctctgcca aattagagac 40380 aggagctggc tgagcccttt ggccaggggaggcagctgtg gggtggagag ggccaggctg 40440 ggctccccaa gtcgagggca tcagctagagttgggccagc cagctgcagc caggcctgct 40500 ggacagcgga tacccagggt ccctgagttctcgcagagaa caagactggt ggggaccccc 40560 tcccagagcc cccagccctc ccagcagccagggtaggggg gagactttgg gggaacagtt 40620 gggggaaagg cccatgccag gacatgcctctgtccaccat gggcctgttg ccactcccac 40680 ggcacagccc cctcctgccg cttcagggagcccggcgtcc gtcttcagca acagatccgt 40740 gcagccccct ctctctgcgc cttgtggtgggaagtgtgct tgggcttcgg ggccctacat 40800 gccctgcctg gcgtggagag aagctcatcccctccctcac tctagagatg ctacctccgt 40860 tgatgaagcc atgggcccca gctccccagcaccgggccgc tacccagtga cacccacccc 40920 tgagccgggt cgaggctcca ccttccctttgttccatctc agggacttgc ctgatgctca 40980 gggctgcctc tgtcccgtcg gagtgccccgtctcccaacc ctgcttcctg cgtctgtcac 41040 ctgggtcggg gtcttgagtc ccctgagcagagtcaggagc tcacggctgg cccaggagct 41100 gctcacgggt gactgggctg ggctggcctgcgggcccgaa ggagactcag aggccagggt 41160 ttggggaaca ggtggcctgt gaagacctttcccggccccc agccctgtgg gcgctgggga 41220 aggtgtcccc ctgtccccgc cttgaagcttccctccatcc ccacagtgtc cctgtcccgg 41280 gagggacttc acgccctttc aggtggggctcacgaggctg aggggggtgc ccagcattca 41340 tgtttgtggt cccctgtgac tgctggcagaggatgatccc gcccggtctg gccaccccca 41400 gagctggtgc ctgcctctgc gaccgctgcctgcccccaga gcttggcaag gaaggagcaa 41460 ctgcctttct ggtgaccgac tcgcttctggtggccggcct cccgtgcagc tgcccctgca 41520 cccgcccact gtgccggccg cccgcacctcgcttgctgat attctctctc tctctgtttc 41580 tgtctgtgcc tctttctgtg tgttctgtagaggacgtagc aaatttaaca gccagtgatg 41640 tcatgaatcg ggtaaacctg ggatatttgcaaggtaattc tgccctggcg ggtgccgacc 41700 accgcacaac ggggctcgcc aacccttcaccgccggcagc cttgctgcgc catcccggcc 41760 aggcaggtgg acaggagctg tgagagcacgtcccaggtgt cagcccaggc ggccctgttg 41820 ggcaaggcct cgcatgccga cagagtgtcccctggctggg cgttgaggac gggcctgagg 41880 ctgaggtctg gggcttgtgc tagtcaaggtgctgttggcc gccaggcaga gtggcgactg 41940 cccagctctc gggctcaccc tggtgctgcttctcctgctt tgctgtttac gatgagctct 42000 gatgggaccg gtgggccacc ctggcctacagccagccctt ccctgtggcc ggccccacaa 42060 gcagtctgag gaccctaagc tcctctgctctcaggaagca cccagagggt cccgagccag 42120 ggccccggga ggagctcggt gaagcctgggctggatgcag gggtggggac tccaagccac 42180 gaggagtagg caggctttat ggctgcccaggccaggtgga tggtgacctt cgtcccccag 42240 ctggggctcc acgaggggat tcttcctgctcggctcgagc agagccgctc cacaccccgt 42300 ttcctccaag aacatgctgt gccccgtcagtgctcagcag agtccgggcc agggtgctcc 42360 caggggtcct ggcagcctca ggggagctcagagcctgccc ccgctgccac acctccctac 42420 agggtaaccc aggagcttga agggcctcctgagccaggac gggtgttagc tcgggaggga 42480 cctgtttgcc acaatgccct tgcacccggaggcagggcca ggcctggggc agccacagaa 42540 gtaaagcgtc ggcctctccc ggctctgtgcttttccccac gatggcgctc atggaagacc 42600 ctcgctctgc cgggcactgt ggtgctcggacctgcagcta gcggccttca tgagtgggat 42660 gcctgcgtcg cccgctcccc tcacccgccaaagcttcctg cggctcccac tgcagctcca 42720 gggcaggcat ggccacagcg ggcttggggggcgagggctc acaggactcc cgcggcaggc 42780 atttcctggg ctcggcctac atgcccgggagggtgacggt gccggggttc tggggctggg 42840 gagctgcacc cccatgttta gattcaagcggagcctaggc caagaggcag aacaggaaga 42900 gtcacaccct ttgcggggag ggcgggcagcatcctggggc ccctccagcg tgcagccccc 42960 atcccacaca cacagcggga ggaggcaccccagccccgat actcacacaa atcccctctt 43020 cacgccttcc tcacagctgc cgtggggggaggcactagtt atccccagtt tatagagaag 43080 gaaactgagg ctcgggtggc ggtagctctggcctggcgtg ggggattcag gcgggagtga 43140 ccgtggatgc caagagccag gaggtctggaagcctctggg gcctgcaggg ttaggaagcg 43200 ggttgggggg cgagcagagc taccactgaggcctggagta aaggtcggag ggtctgggtg 43260 gaggagtcag gtagccctgg ggaccaggggcgaggtggcc ctgccccagg acatgcacca 43320 gaagagccaa agccagcccc tgccccaccccaggccctca ggcacacctg ctgccagcct 43380 tcaccaagca ccgtggacac gcgaagctagtcctctgcga ccatcctggg cagccaggac 43440 cagtgccatg tgactgacca caacccaggccctggcactg cagctggggc ctggcatccc 43500 cacccgcccc ctagcccagc cccaagaaacacaaagccca gcctggctca gcccagtgag 43560 ctctgcttac ttcagaagcg tgtggtccgggcaccccagc gggagaagct ggggcctggg 43620 ggccagtctg tgcctgccgg agtgaaatgagctggtggcc ccttgttctc ggtgtcccac 43680 tgggtgctcg tcacaaagcc agtcaggcaggggggcaggg tcccaagaac ctactagagc 43740 tgccctcacc acccactccc tgtgccacctgccatgcggc caccccagaa gcgtgtgtgg 43800 gcacgagggt ggggagggtg tgtctccctgcacagcctca gcatgaggcc cctgggcctc 43860 ctccaaaggc ctggggaaga ccccaccatgggttttggga aaagagcaaa acagctgtga 43920 ggggcaggtg tcaggccgtc ccatcccgcaggtgtggaca caggagctgg caccaaatga 43980 cctgccgccg tgtgcgaaag gcctgaagcagagctggtct cctggccgga gctgccctgg 44040 tcgcccccaa gtcctagcct gtgcccccgccaccctggca gcagctgtgc tcacactgtg 44100 ggaactccaa agcccccact gcagagggaccagcatggcc cccacctggc tggccagcag 44160 cctccccgac ttttcccttc tcagggaccagggcagggct accccaaggg gagaggacgg 44220 gcagagagga gggaatggca gcagccagggtggaggctcc ctggggtccc ccgtcctggg 44280 tcacctttgt tctctgtcct tggagtcctggaacaggaat ccaggagctt cccgtagaca 44340 ccatcagatg gataaactga ggccacagaccctttccagc cagtgaaccc agggccactg 44400 ctgggatggt ggcctctccc tccctgtgcttggcagggct accctgccgg ccccatggag 44460 actgtgagat gatggggggg ctgtctctccaggaccgttc tcaccctgtg ggacacatgc 44520 ccacgcccac cccatgagcc ggacgccaggctgtctccca tgaggggcag aggccatgtt 44580 ctcggaggga tgagctgcgg ggtgggggtgctctgggctg atgccactca ggacttcagt 44640 ctgaagcaaa gggctgagca cagcgagtgggaatggcctg tccaggcccg gggacagcgg 44700 cagctgcagg gccctgaggg agggacagggctgagcagcc tctggaggcg ggggtggggg 44760 tgtgtggatg tggaggacgg catgcaggtgtgtatatgag tgagtgtgca ggggtctgtg 44820 gataccatag gcatgggttg ctatgaagatctgtgcagac acacaaatgt gtgtggaata 44880 cagaggtgaa tctgcaggtg tgtgcaggtgtagatgaagg catgcatgca tgtgaggtga 44940 acagacacgt cccggtgtct gcaggctgacgtacgtggca ggtgtgcgct ggtgtgtgca 45000 ggtgtgctca gtgcctctgc tcagggccacccctttgctg cgatgccacc tagcaccaga 45060 ggtgggtgcg ggtcagagct ggctcccagctgccctcctg gctgtcccct gccctgtgtg 45120 gagatgggac aggtgtcggt cagggatgggccctggctgg gctcaccagg atgaggtgct 45180 ggagcagaat ggtcaggaag gccggaaagactcagctcat cctggagccc ccacacctct 45240 gggcccctgc agagctggga ggggcagggctgggggggtg acgtctgccc ggctgtgtcc 45300 tttgcagacg agatgaacga ccaccagaacaccctctcct acgtcctcat caaccctccg 45360 cccgacacga ggctggagcc cagtgacattgtgtgagtag cacctgtggg ctgtgtggag 45420 accccccctg agcaccaggt gggcactggggagatgaggc cacaggcacc acagtggggc 45480 cgctcagcag agggctgagc aggggctgcccggcccacat ccactccagg gtcctctgtg 45540 ccctcccgca gctatctcat ccgctccgaccccctggctc acgtggccag cagctcccag 45600 agccggaaga gcagctgcag ccacaagctgtcgtcctgca accccgagac tcgcgacgag 45660 acacagctct gagccagccc tgcacggagctcaggccacc aagcccgggg tcctcaggaa 45720 ggacgtggag gagcgtgtga ggacacggtggcactagcgt gaccctgggg atggcacact 45780 ctactcacca tggctcctgg gactccaccctggaaaggag cccctcatgc ggggggaggg 45840 ccagctcacc cctgggcacc tgcaggctagtgaggagagt tttttaacct atttttacac 45900 gtcgatgcag tccacttctc tttacacagatgtaccgcaa ctcgtgacca gggctggctg 45960 ggagggcaac gcagggactg gacgccctacagggccgagc ccaggctgtg ctggagggtg 46020 gggctggggt gcatggggag gggagcagaacccagaaccc aggagccccg cgtgggccac 46080 acccaactca gagccggcct gagcgttcacggccaggcag cctcgcttcc ttgcagccaa 46140 gggctggggg ccagggctgc tgttctgcactctggggtgg gtgaggggga ccctgggctg 46200 tttgctgtcc caagcccctt ctggaagttagaagcagcaa agggcccggg gaagccgggc 46260 atgtgagagg ggtgcgtccc caggtcccccagagggccct gtcgccgagg acctttctga 46320 aggaagcaga agacgccatt tcctctacttcacactgaac tgtcccagcc actgcatcta 46380 gggggcattg ggcggaagat ggtgcatttccatggaccat tttacactta ccttttaaag 46440 caaagcctca ttttctaaac ccctgacttgtgaagcacaa ttcagcctcc gggctgggcc 46500 acgtggagag agaggatctt ctcagcaaggcgagatcccg ggcggcggct gacatcagga 46560 gcgccaccct gcgtcctttg ctgctggttccttactggtt tgtacggtca gcgctggaaa 46620 cttctattaa atggatgcat tctggaggcatgaagttaca agtcaagtcg cccaattaaa 46680 tgtcacactg cccagccctg cactcctctcccacttggcc agtttcaggc tgtaccactc 46740 caccgaggtg accccagcta gtttggggcttcagatccca ctgctgagcc ggagacccag 46800 atgggtctca gactgacagt ctcagactggcagcccccca tagcaccagg cgtgggtgcc 46860 cacccacgag atgtcacctg gcaggcactttgagaatgtc cagaatgggt gtcataatcc 46920 accccccgcc tgctcctttt gtcttccccaccttggggtg tcaccacctg cctgttccct 46980 ccccccagcc tgggcgctgt ccctcccccctagtccttca ggaagtctag ccagtgccac 47040 cttcaaaaca caccttggat ccacctggtcccctgtcctc tgtccagccc gcagtcccac 47100 tcagctcgcg gtaggcaaag cgaggccccaccctctctgc agggctcttg gagacacgtt 47160 tgaaccacag cgtctgtgtg ctctctaaaagggcagttgt tattttttcc tgagtttcca 47220 ggatgtctgc ccctgtgtag ccatagggtcgagcctctcc aggctccatg ggagcagaaa 47280 gtgctgggga cacctcaagg ctcacgtggctctgtggacc ccgagcctgg ggccccagcc 47340 caccttcccc tccgtgcctg gctccagccatgggccctgc acccgtcctg gtgccctgag 47400 gccaggaaag ggggcttctg ggactgctgctcgatgccga aggtattgtc cttcccacat 47460 gggaatcatc tcactggact ttctgtgacaaacatagaca gtcatttcaa aataaatcaa 47520 acttcagacg tggtacagca gcaagttgcgaagatcctcc tcccctcccc gcaccccagg 47580 cggcacctgc tgcagccggc tgtgggcactccatggacaa cacggcacag atgttctctt 47640 gtcacataac tctcatcctg tacttcaaaggccacgagta agtgaagacc cactgcactc 47700 tcgaaaagca ggaacggaaa aagtcacagcactgcatcat ggagcagccg ccccgggtgg 47760 gcgcgcgcgt ttgcctcctg cgtgaggcttcctccagcac tgcggtgggc tgtgggttcg 47820 ttcccaggga atgtgagcga gccgcatccgcagcccaggc accctccctc ccagcttcct 47880 ccccgcccca gggtgaccat gtatgggcagccctgatgtc catcggccct ttgtgcagtc 47940 atggcgcggt gaagggccct gcaggcggcgcccaggggtg gtgcggggct ccgcctggtc 48000 tgtttactca cagcacagca tggggctcactcagcagcac gcccaggaga agagcccagc 48060 ctcattcttt acagccgttc tggggcactccacaggaggg ttgaaggggg cttccatctg 48120 ctggtgctcc ccaacaccag gggctggcagagggtactcg tgctttgcag gtacaaacag 48180 cgctctcccc tggggtagac ccgagccacgacaggcagcc agggcgacat gcataggagc 48240 gtgtgctcag accacacaca gtggcttttcaggggatttt ccttactcag ccacatcggg 48300 gagggcgcta gggcaggtat cgggagcgccagtgagttgg tcattttctg agcatgatgg 48360 cggctcagcg tttggacccc cagagggagcagggccctgt gcctcagcct ccctcgagtg 48420 gtccagccct ccctcagcat gggtgctgaagaaatggaag cctgtgctgc ctgtcaggga 48480 cctgccgctg gagttttacc cccaccccatccgcgttggc cacccaggcc cctgtggcac 48540 gggtgcatgg aggcctagtg aggtcagaaaccgtccagcc aagggtgggg ggattcaggg 48600 tgggggaaca ggcagccggc tccgggagggcttctggtgt gagtttgggg cgggccggcc 48660 cactccc 48667 4 1170 PRT Rattusnorvegicus 4 Val Gln Val Glu Phe Tyr Val Asn Glu Asn Thr Phe Lys Glu ArgLeu 1 5 10 15 Lys Leu Phe Phe Ile Lys Asn Gln Arg Ser Ser Leu Arg IleArg Leu 20 25 30 Phe Asn Phe Ser Leu Lys Leu Leu Thr Cys Leu Leu Tyr IleVal Arg 35 40 45 Val Leu Leu Asp Asn Pro Asp Gln Gly Ile Gly Cys Trp GlyCys Thr 50 55 60 Lys Tyr Asn Tyr Thr Phe Asn Gly Ser Ser Ser Glu Phe HisTrp Ala 65 70 75 80 Pro Ile Leu Trp Val Glu Arg Lys Met Ala Leu Trp ValIle Gln Val 85 90 95 Ile Val Ala Thr Ile Ser Phe Leu Glu Thr Met Leu LeuIle Tyr Leu 100 105 110 Ser Tyr Lys Gly Asn Ile Trp Glu Gln Ile Phe HisVal Ser Phe Val 115 120 125 Leu Glu Met Ile Asn Thr Leu Pro Phe Ile IleThr Val Phe Trp Pro 130 135 140 Pro Leu Arg Asn Leu Phe Ile Pro Val PheLeu Asn Cys Trp Leu Ala 145 150 155 160 Lys His Ala Leu Glu Asn Met IleAsn Asp Phe His Arg Ala Ile Leu 165 170 175 Arg Thr Gln Ser Ala Met PheAsn Gln Val Leu Ile Leu Phe Cys Thr 180 185 190 Leu Leu Cys Leu Val PheThr Gly Thr Cys Gly Ile Gln His Leu Glu 195 200 205 Arg Ala Gly Gly AsnLeu Asn Leu Leu Thr Ser Phe Tyr Phe Cys Ile 210 215 220 Val Thr Phe SerThr Val Gly Phe Gly Asp Val Thr Pro Lys Ile Trp 225 230 235 240 Pro SerGln Leu Leu Val Val Ile Leu Ile Cys Val Thr Leu Val Val 245 250 255 LeuPro Leu Gln Phe Glu Glu Leu Val Tyr Leu Trp Met Glu Arg Gln 260 265 270Lys Ser Gly Gly Asn Tyr Ser Arg His Arg Ala Arg Thr Glu Lys His 275 280285 Val Val Leu Cys Val Ser Ser Leu Lys Ile Asp Leu Leu Met Asp Phe 290295 300 Leu Asn Glu Phe Tyr Ala His Pro Arg Leu Gln Asp Tyr Tyr Val Val305 310 315 320 Ile Leu Cys Pro Ser Glu Met Asp Val Gln Val Arg Arg ValLeu Gln 325 330 335 Ile Pro Leu Trp Ser Gln Arg Val Ile Tyr Leu Gln GlySer Ala Leu 340 345 350 Lys Asp Gln Asp Leu Met Arg Ala Lys Met Asp AsnGly Glu Ala Cys 355 360 365 Phe Ile Leu Ser Ser Arg Asn Glu Val Asp ArgThr Ala Ala Asp His 370 375 380 Gln Thr Ile Leu Arg Ala Trp Ala Val LysAsp Phe Ala Pro Asn Cys 385 390 395 400 Pro Leu Tyr Val Gln Ile Leu LysPro Glu Asn Lys Phe His Val Lys 405 410 415 Phe Ala Asp His Val Val CysGlu Glu Glu Cys Lys Tyr Ala Met Leu 420 425 430 Ala Leu Asn Cys Ile CysPro Ala Thr Ser Thr Leu Ile Thr Leu Leu 435 440 445 Val His Thr Ser ArgGly Gln Glu Gly Gln Glu Ser Pro Glu Gln Trp 450 455 460 Gln Arg Met TyrGly Arg Cys Ser Gly Asn Glu Val Tyr His Ile Arg 465 470 475 480 Met GlyAsp Ser Lys Phe Phe Arg Glu Tyr Glu Gly Lys Ser Phe Thr 485 490 495 TyrAla Ala Phe His Ala His Lys Lys Tyr Gly Val Cys Leu Ile Gly 500 505 510Leu Lys Arg Glu Glu Asn Lys Ser Ile Leu Leu Asn Pro Gly Pro Arg 515 520525 His Ile Leu Ala Ala Ser Asp Thr Cys Phe Tyr Ile Asn Ile Thr Lys 530535 540 Glu Glu Asn Ser Ala Phe Ile Phe Lys Gln Glu Glu Lys Gln Asn Arg545 550 555 560 Arg Gly Leu Ala Gly Gln Ala Leu Tyr Glu Gly Pro Ser ArgLeu Pro 565 570 575 Val His Ser Ile Ile Ala Ser Met Val Ala Met Asp LeuGln Asn Thr 580 585 590 Asp Cys Arg Pro Ser Gln Gly Gly Ser Gly Gly GlyGly Gly Lys Leu 595 600 605 Thr Leu Pro Thr Glu Asn Gly Ser Gly Ser ArgArg Pro Ser Ile Ala 610 615 620 Pro Val Leu Glu Leu Ala Asp Ser Ser AlaLeu Leu Pro Cys Asp Leu 625 630 635 640 Leu Ser Asp Gln Ser Glu Asp GluVal Thr Pro Ser Asp Asp Glu Gly 645 650 655 Leu Ser Val Val Glu Tyr ValLys Gly Tyr Pro Pro Asn Ser Pro Tyr 660 665 670 Ile Gly Ser Ser Pro ThrLeu Cys His Leu Leu Pro Val Lys Ala Pro 675 680 685 Phe Cys Cys Leu ArgLeu Asp Lys Gly Cys Lys His Asn Ser Tyr Glu 690 695 700 Asp Ala Lys AlaTyr Gly Phe Lys Asn Lys Leu Ile Ile Val Ser Ala 705 710 715 720 Glu ThrAla Gly Asn Gly Leu Tyr Asn Phe Ile Val Pro Leu Arg Ala 725 730 735 TyrTyr Arg Ser Arg Arg Glu Leu Asn Pro Ile Val Leu Leu Leu Asp 740 745 750Asn Lys Pro Asp His His Phe Leu Glu Ala Ile Cys Cys Phe Pro Met 755 760765 Val Tyr Tyr Met Glu Gly Ser Val Asp Asn Leu Asp Ser Leu Leu Gln 770775 780 Cys Gly Ile Ile Tyr Ala Asp Asn Leu Val Val Val Asp Lys Glu Ser785 790 795 800 Thr Met Ser Ala Glu Glu Asp Tyr Met Ala Asp Ala Lys ThrIle Val 805 810 815 Asn Val Gln Thr Met Phe Arg Leu Phe Pro Ser Leu SerIle Thr Thr 820 825 830 Glu Leu Thr His Pro Ser Asn Met Arg Phe Met GlnPhe Arg Ala Lys 835 840 845 Asp Ser Tyr Ser Leu Ala Leu Ser Lys Leu GluLys Gln Glu Arg Glu 850 855 860 Asn Gly Ser Asn Leu Ala Phe Met Phe ArgLeu Pro Phe Ala Ala Gly 865 870 875 880 Arg Val Phe Ser Ile Ser Met LeuAsp Thr Leu Leu Tyr Gln Ser Phe 885 890 895 Val Lys Asp Tyr Met Ile ThrIle Thr Arg Leu Leu Leu Gly Leu Asp 900 905 910 Thr Thr Pro Gly Ser GlyTyr Leu Cys Ala Met Lys Val Thr Glu Asp 915 920 925 Asp Leu Trp Ile ArgThr Tyr Gly Arg Leu Phe Gln Lys Leu Cys Ser 930 935 940 Ser Ser Ala GluIle Pro Ile Gly Ile Tyr Arg Thr Glu Cys His Val 945 950 955 960 Phe SerSer Glu Pro His Asp Leu Arg Ala Gln Ser Gln Ile Ser Val 965 970 975 AsnMet Glu Asp Cys Glu Asp Thr Arg Glu Ala Lys Gly Pro Trp Gly 980 985 990Thr Arg Ala Ala Ser Gly Gly Gly Ser Thr His Gly Arg His Gly Gly 995 10001005 Ser Ala Asp Pro Val Glu His Pro Leu Leu Arg Arg Lys Ser Leu Gln1010 1015 1020 Trp Ala Arg Lys Leu Ser Arg Lys Ser Ser Lys Gln Ala GlyLys Ala 1025 1030 1035 1040 Pro Met Thr Thr Asp Trp Ile Thr Gln Gln ArgLeu Ser Leu Tyr Arg 1045 1050 1055 Arg Ser Glu Arg Gln Glu Leu Ser GluLeu Val Lys Asn Arg Met Lys 1060 1065 1070 His Leu Gly Leu Pro Thr ThrGly Tyr Glu Asp Val Ala Asn Leu Thr 1075 1080 1085 Ala Ser Asp Val MetAsn Arg Val Asn Leu Gly Tyr Leu Gln Asp Glu 1090 1095 1100 Met Asn AspHis His Gln Asn Thr Leu Ser Tyr Val Leu Ile Asn Pro 1105 1110 1115 1120Pro Pro Asp Thr Arg Leu Glu Pro Asn Asp Ile Val Tyr Leu Ile Arg 11251130 1135 Ser Asp Pro Leu Ala His Val Thr Ser Ser Ser Gln Ser Arg LysSer 1140 1145 1150 Ser Cys Ser Asn Lys Leu Ser Ser Cys Asn Pro Glu ThrArg Asp Glu 1155 1160 1165 Thr Gln 1170 5 1044 PRT HUMAN 5 Val Gln ValGlu Phe Tyr Val Asn Glu Asn Thr Phe Lys Glu Arg Leu 1 5 10 15 Lys LeuPhe Phe Ile Lys Asn Gln Arg Ser Ser Leu Arg Ile Arg Leu 20 25 30 Phe AsnPhe Ser Leu Lys Leu Leu Thr Cys Leu Leu Tyr Ile Val Arg 35 40 45 Val LeuLeu Asp Asp Pro Ala Leu Gly Ile Gly Cys Trp Gly Cys Pro 50 55 60 Lys GlnAsn Tyr Ser Phe Asn Asp Ser Ser Ser Glu Ile Asn Trp Ala 65 70 75 80 ProIle Leu Trp Val Glu Arg Lys Met Thr Leu Trp Ala Ile Gln Val 85 90 95 IleVal Ala Ile Ile Ser Phe Leu Glu Thr Met Leu Leu Ile Tyr Leu 100 105 110Ser Tyr Lys Gly Asn Ile Trp Glu Gln Ile Phe Arg Val Ser Phe Val 115 120125 Leu Glu Met Ile Asn Thr Leu Pro Phe Ile Ile Thr Ile Phe Trp Pro 130135 140 Pro Leu Arg Asn Leu Phe Ile Pro Val Phe Leu Asn Cys Trp Leu Ala145 150 155 160 Lys His Ala Leu Glu Asn Met Ile Asn Asp Phe His Arg AlaIle Leu 165 170 175 Arg Thr Gln Ser Ala Met Phe Asn Gln Val Leu Ile LeuPhe Cys Thr 180 185 190 Leu Leu Cys Leu Val Phe Thr Gly Thr Cys Gly IleGln His Leu Glu 195 200 205 Arg Ala Gly Glu Asn Leu Ser Leu Leu Thr SerPhe Tyr Phe Cys Ile 210 215 220 Val Thr Phe Ser Thr Val Gly Tyr Gly AspVal Thr Pro Lys Ile Trp 225 230 235 240 Pro Ser Gln Leu Leu Val Val IleMet Ile Cys Val Ala Leu Val Val 245 250 255 Leu Pro Leu Gln Phe Glu GluLeu Val Tyr Leu Trp Met Glu Arg Gln 260 265 270 Lys Ser Gly Gly Asn TyrSer Arg His Arg Ala Gln Thr Glu Lys His 275 280 285 Val Val Leu Cys ValSer Ser Leu Lys Ile Asp Leu Leu Met Asp Phe 290 295 300 Leu Asn Glu PheTyr Ala His Pro Arg Leu Gln Asp Tyr Tyr Val Val 305 310 315 320 Ile LeuCys Pro Thr Glu Met Asp Val Gln Val Arg Arg Val Leu Gln 325 330 335 IlePro Leu Trp Ser Gln Arg Val Ile Tyr Leu Gln Gly Ser Ala Leu 340 345 350Lys Asp Gln Asp Leu Met Arg Ala Lys Met Asp Asn Gly Glu Ala Cys 355 360365 Phe Ile Leu Ser Ser Arg Asn Glu Val Asp Arg Thr Ala Ala Asp His 370375 380 Gln Thr Ile Leu Arg Ala Trp Ala Val Lys Asp Phe Ala Pro Asn Cys385 390 395 400 Pro Leu Tyr Val Gln Ile Leu Lys Pro Glu Asn Lys Phe HisVal Lys 405 410 415 Phe Ala Asp His Val Val Cys Glu Glu Glu Cys Lys TyrAla Met Leu 420 425 430 Ala Leu Asn Cys Ile Cys Pro Ala Thr Ser Thr LeuIle Thr Leu Leu 435 440 445 Val His Thr Ser Arg Gly Gln Glu Gly Gln GluSer Pro Glu Gln Trp 450 455 460 Gln Arg Met Tyr Gly Arg Cys Ser Gly AsnGlu Val Tyr His Ile Arg 465 470 475 480 Met Gly Asp Ser Lys Phe Phe ArgGlu Tyr Glu Gly Lys Ser Phe Thr 485 490 495 Tyr Ala Ala Phe His Ala HisLys Lys Tyr Gly Val Cys Leu Ile Gly 500 505 510 Leu Lys Arg Glu Asp AsnLys Ser Ile Leu Leu Asn Pro Gly Pro Arg 515 520 525 His Ile Leu Ala AlaSer Asp Thr Cys Phe Tyr Ile Asn Ile Thr Lys 530 535 540 Glu Glu Asn SerAla Phe Ile Phe Lys Gln Glu Glu Lys Arg Lys Lys 545 550 555 560 Arg AlaPhe Ser Gly Gln Gly Leu His Glu Gly Pro Ala Arg Leu Pro 565 570 575 ValHis Ser Ile Ile Ala Ser Met Gly Thr Val Ala Met Asp Leu Gln 580 585 590Gly Thr Glu His Arg Pro Thr Gln Ser Gly Gly Gly Gly Gly Gly Ser 595 600605 Lys Leu Ala Leu Pro Thr Glu Asn Gly Ser Gly Ser Arg Arg Pro Ser 610615 620 Ile Ala Pro Val Leu Glu Leu Ala Asp Ser Ser Ala Leu Leu Pro Cys625 630 635 640 Asp Leu Leu Ser Asp Gln Ser Glu Asp Glu Val Thr Pro SerAsp Asp 645 650 655 Glu Gly Leu Ser Val Val Glu Tyr Val Lys Gly Tyr ProPro Asn Ser 660 665 670 Pro Tyr Ile Gly Ser Ser Pro Thr Leu Cys His LeuLeu Pro Val Lys 675 680 685 Ala Pro Phe Cys Cys Leu Arg Leu Asp Lys GlyCys Lys His Asn Ser 690 695 700 Tyr Glu Asp Ala Lys Ala Tyr Gly Phe LysAsn Lys Leu Ile Ile Val 705 710 715 720 Ser Ala Glu Thr Ala Gly Asn GlyLeu Tyr Asn Phe Ile Val Pro Leu 725 730 735 Arg Ala Tyr Tyr Arg Ser ArgLys Glu Leu Asn Pro Ile Val Leu Leu 740 745 750 Leu Asp Asn Lys Pro AspHis His Phe Leu Glu Ala Ile Cys Cys Phe 755 760 765 Pro Met Val Tyr TyrMet Glu Gly Ser Val Asp Asn Leu Asp Ser Leu 770 775 780 Leu Gln Cys GlyIle Ile Tyr Ala Asp Asn Leu Val Val Val Asp Lys 785 790 795 800 Glu SerThr Met Ser Ala Glu Glu Asp Tyr Met Ala Asp Ala Lys Thr 805 810 815 IleVal Asn Val Gln Thr Met Phe Arg Leu Phe Pro Ser Leu Ser Ile 820 825 830Thr Thr Glu Leu Thr His Pro Ser Asn Met Arg Phe Met Gln Phe Arg 835 840845 Ala Lys Asp Ser Tyr Ser Leu Ala Leu Ser Lys Leu Glu Lys Arg Glu 850855 860 Arg Glu Asn Gly Ser Asn Leu Ala Phe Met Phe Arg Leu Pro Phe Ala865 870 875 880 Ala Gly Arg Val Phe Ser Ile Ser Met Leu Asp Thr Leu LeuTyr Gln 885 890 895 Ser Phe Val Lys Asp Tyr Met Ile Thr Ile Thr Arg LeuLeu Leu Gly 900 905 910 Leu Asp Thr Thr Pro Gly Ser Gly Tyr Leu Cys AlaMet Lys Ile Thr 915 920 925 Glu Gly Asp Leu Trp Ile Arg Thr Tyr Gly ArgLeu Phe Gln Lys Leu 930 935 940 Cys Ser Ser Ser Ala Glu Ile Pro Ile GlyIle Tyr Arg Thr Glu Ser 945 950 955 960 His Val Phe Ser Thr Ser Glu ProHis Asp Leu Arg Ala Gln Ser Gln 965 970 975 Ile Ser Val Asn Val Glu AspCys Glu Asp Thr Arg Glu Val Lys Gly 980 985 990 Pro Trp Gly Ser Arg AlaGly Thr Gly Gly Ser Ser Gln Gly Arg His 995 1000 1005 Thr Gly Gly GlyAsp Pro Ala Glu His Pro Leu Leu Arg Arg Lys Ser 1010 1015 1020 Leu GlnTrp Ala Arg Arg Leu Ser Arg Lys Ala Pro Lys Gln Ala Gly 1025 1030 10351040 Arg Ala Ala Ala

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human transporter protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human transporter peptide having an aminoacid sequence that shares at least 70% homology with an amino acidsequence shown in SEQ ID NO:2.
 21. A peptide according to claim 20 thatshares at least 90 percent homology with an amino acid sequence shown inSEQ ID NO:2.
 22. An isolated nucleic acid molecule encoding a humantransporter peptide, said nucleic acid molecule sharing at least 80percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or3.
 23. A nucleic acid molecule according to claim 22 that shares atleast 90 percent homology with a nucleic acid molecule shown in SEQ IDNOS:1 or 3.